WO2018213731A1 - Polynucléotides codant pour des polypeptides d'interleukine-12 (il12) ancrés et leurs utilisations - Google Patents

Polynucléotides codant pour des polypeptides d'interleukine-12 (il12) ancrés et leurs utilisations Download PDF

Info

Publication number
WO2018213731A1
WO2018213731A1 PCT/US2018/033436 US2018033436W WO2018213731A1 WO 2018213731 A1 WO2018213731 A1 WO 2018213731A1 US 2018033436 W US2018033436 W US 2018033436W WO 2018213731 A1 WO2018213731 A1 WO 2018213731A1
Authority
WO
WIPO (PCT)
Prior art keywords
polynucleotide
mrna
polypeptide
seq
domain
Prior art date
Application number
PCT/US2018/033436
Other languages
English (en)
Inventor
Ankita MISHRA
Joshua Frederick
Sushma GURUMURTHY
Original Assignee
Modernatx, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Modernatx, Inc. filed Critical Modernatx, Inc.
Priority to JP2019563454A priority Critical patent/JP7285220B2/ja
Priority to US16/613,732 priority patent/US11421011B2/en
Priority to CA3063723A priority patent/CA3063723A1/fr
Priority to EP18729295.8A priority patent/EP3625246A1/fr
Priority to AU2018270111A priority patent/AU2018270111B2/en
Publication of WO2018213731A1 publication Critical patent/WO2018213731A1/fr
Priority to US17/875,887 priority patent/US11873327B2/en
Priority to JP2023015202A priority patent/JP2023059880A/ja

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • Interleukin-12 is a pro-inflammatory cytokine that plays an important role in innate and adaptive immunity. Gately, MK et al., Annu Rev Immunol. 16: 495-521 (1998). IL-12 functions primarily as a 70 kDa heterodimeric protein consisting of two disulfide-linked p35 and p40 subunits. IL-12 p40 homodimers do exist, but other than functioning as an antagonist that binds the IL-12 receptor, they do not appear to mediate a biologic response. Id.
  • the precursor form of the IL-12 p40 subunit (NM_002187; P29460; also referred to as IL-12B, natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2) is 328 amino acids in length, while its mature form is 306 amino acids long.
  • the precursor form of the IL-12 p35 subunit (NM_000882; P29459; also referred to as IL-12A, natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1) is 219 amino acids in length and the mature form is 197 amino acids long. Id.
  • the genes for the IL-12 p35 and p40 subunits reside on different chromosomes and are regulated independently of each other.
  • IL-12 protein Due to its ability to activate both NK cells and cytotoxic T cells, IL-12 protein has been studied as a promising anti-cancer therapeutic since 1994. See Nastala, C. L. et al., J Immunol 153: 1697-1706 (1994). But despite high expectations, early clinical studies did not yield satisfactory results. Lasek W. et al., Cancer Immunol Immunother 63: 419-435, 424 (2014). Repeated
  • IFNy interferon gamma
  • the marginal efficacy of the IL-12 therapy in clinical settings may be caused by the strong immunosuppressive environment in humans.
  • Id To minimize IFNy toxicity and improve IL-12 efficacy, scientists tried different approaches, such as different dose and time protocols for IL-12 therapy. See Sacco, S. et al, Blood 90: 4473-4479 (1997); Leonard, J. P. et al, Blood 90: 2541-2548 (1997); Coughlin, C. M. et al, Cancer Res. 57: 2460-2467 (1997); Asselin-Paturel, C. et al., Cancer 91: 113-122 (2001); and Saudemont, A. et al, Leukemia 16: 1637-1644 (2002). Nonetheless, these approaches have not significantly impacted patient survival. Kang, W. K., et al., Human Gene Therapy 12: 671-684 (2001).
  • Membrane- anchored versions of IL-12 have been studied as a means of reducing toxicity associated with systemic administration, using retroviral and adenoviral vectors for expression in tumor cells. See Pan, W-Y. et al, Mol. Ther. 20(5): 927-937 (2012). But, the use of viral vectors presents a potential health risk, since the underlying viruses can act as oncogenes and the viral vectors can be immunogenic.
  • the present disclosure is directed to novel tethered interleukin-12 (IL-12)-encoding polynucleotides (e.g., mRNAs) for use in treating cancer.
  • IL-12 interleukin-12
  • IL-12 has been shown to have potent anti-tumor activity, its clinical application is limited by severe systemic toxicity. Several strategies have been employed to address this limitation, which appear promising.
  • the present disclosure is based, at least in part, on a strategy of anchoring an IL-12 polypeptide to a cell membrane by delivering an mRNA encoding an IL-12 polypeptide to the cell, thereby generating a tethered IL-12 polypeptide with reduce systemic distribution. Further, the present disclosure is based on the discovery that tethered IL-12
  • polypeptides encoded by mRNA, remain substantially tethered to the cell surface (i.e., are not substantially released by cells expressing the mRNA encoded IL-12 polypeptide), thereby reducing systemic distribution. It has also been discovered that mRNA encoded tethered IL-12 polypeptides retain IL-12 bioactivity. Specifically, mRNA encoding a tethered IL-12 polypeptide as described herein was shown to induce an anti-tumor immune response, as indicated by an increase in CD8+ T cell proliferation and IFNy secretion, along with a reduction in tumor burden in vivo in both treated and non-treated (i.e., distal) tumors.
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) comprising: (a) a first nucleic acid sequence encoding an Interleukin 12 p40 subunit (IL-12B), (b) a second nucleic acid sequence encoding an Interleukin 12 p35 subunit (IL- 12A), and (c) a nucleic acid sequence encoding a transmembrane domain, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a nucleic acid sequence encoding a linker ("subunit linker"), and wherein the nucleic acid sequence encoding the transmembrane domain is linked to the first or second nucleic acid sequence by a nucleic acid sequence encoding a linker ("transmembrane domain linker").
  • ORF open reading frame
  • the first nucleic acid sequence is located at the 5' end of the subunit linker.
  • the nucleic acid sequence encoding the transmembrane domain is located at the 3' end of the transmembrane domain linker.
  • the polynucleotide further comprises a nucleic acid sequence encoding a signal peptide.
  • the nucleic acid sequence encoding the signal peptide is located at the 5' end of the first nucleic acid sequence.
  • the IL-12B has an amino acid sequence at least about 80%, at least about 90%, at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 23 to 328 of SEQ ID NO: 48, and wherein the amino acid sequence has IL- 12B activity.
  • the IL-12A has an amino acid sequence at least about 80%, at least about 90%, at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 336 to 532 of SEQ ID NO: 48, and wherein the amino acid sequence has IL- 12A activity.
  • the signal peptide comprises a sequence at least about 80%, at least about 90%, at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 1 to 22 of SEQ ID NO: 48.
  • the subunit linker is a Gly/Ser linker.
  • the transmembrane domain linker is a Gly/Ser linker.
  • the Gly/Ser linker comprises (G n S) m , wherein n is 1, 2 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20.
  • the transmembrane domain is a type I transmembrane domain. In some embodiments, the transmembrane domain is a Cluster of Differentiation 8 (CD8) transmembrane domain or a Platelet-Derived Growth Factor Receptor (PDGF-R) transmembrane domain.
  • CD8 Cluster of Differentiation 8
  • PDGF-R Platelet-Derived Growth Factor Receptor
  • the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the polynucleotide is mRNA.
  • the polynucleotide comprises at least one chemically modified nucleobase.
  • the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil ( ⁇ ), Nl-methylpseudouracil 2-thiouracil (s2U), 4'- thiouracil, 5-methylcytosine, 2-thio-l -methyl- 1-deaza-pseudouracil, 2-thio-l-methyl-pseudouracil, 2-thio-5-aza-uracil, 2-thio-dihydropseudouracil, 2-thio-dihydrouracil, 2-thio-pseudouracil, 4- methoxy-2-thio-pseudouracil, 4-methoxy-pseudouracil, 4-thio-l -methyl -pseudouracil, 4-thio- pseudouracil, 5-aza-uracil, dihydropseudouracil, 5-methyluracil, 5-methoxyuracil, 2' -O-methyl
  • the nucleobases in the polynucleotide are chemically modified by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 95%, at least 99%, or 100%.
  • the chemically modified nucleobases are selected from the group consisting of uracil, adenine, cytosine, guanine, and any combination thereof.
  • the uracils, adenines, cytosines or guanines are chemically modified by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 95%, at least 99%, or 100%.
  • the polynucleotide further comprises a nucleic acid sequence comprising a miRNA binding site.
  • the miRNA binding site binds to miR-122. In some embodiments, the miRNA binding site binds to miR-122-3p or miR-122-5p.
  • the polynucleotide further comprises a 5' UTR.
  • the 5' UTR comprises a nucleic acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of sequences disclosed herein.
  • the polynucleotide further comprises a 3' UTR.
  • the 3' UTR comprises a nucleic acid sequence at least about 90%, at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of sequences disclosed herein.
  • the miRNA binding site is located within the 3' UTR.
  • the 5' UTR comprises a 5' terminal cap.
  • the 5' terminal cap is a CapO, Capl, ARCA, inosine, Nl-methyl- guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof.
  • the polynucleotide further comprises a poly-A region.
  • the poly-A region is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, or at least about 90 nucleotides in length.
  • the poly-A region has about 10 to about 200 nucleotides in length, about 20 to about 180 nucleotides in length, about 30 to about 160 nucleotides in length, about 40 to about 140 nucleotides in length, about 50 to about 120 nucleotides in length, about 60 to about 100 nucleotides in length, or about 80 to about 90 nucleotides in length.
  • the polynucleotide has been transcribed in vitro (IVT). In some embodiments, the polynucleotide is chimeric. In some embodiments, the polynucleotide is circular.
  • the ORF further comprises one or more nucleic acid sequences encoding one or more heterologous polypeptides fused to the nucleic acid sequence encoding the IL- 12B, the IL-12A, or both.
  • the one or more heterologous polypeptides increase a pharmacokinetic property of the IL-12A, the IL-12B, or both.
  • the polynucleotide is single stranded. In some embodiments, the polynucleotide is double stranded. In some embodiments, the IL-12B is a variant, derivative or mutant having an IL-12B activity. In some embodiments, the IL-12A is a variant, derivative, or mutant having an IL-12A activity. In another aspect, the disclosure provides a vector comprising any of the above
  • the disclosure provides a composition comprising (i) any of the above polynucleotides or vector, and (ii) a delivery agent.
  • the delivery agent comprises a lipid nanoparticle.
  • the lipid nanoparticle comprises the compound of formula (I).
  • the delivery agent further comprises a phospholipid.
  • the delivery agent further comprises a structural lipid.
  • the structural lipid is cholesterol.
  • the delivery agent further comprises a PEG lipid.
  • the delivery agent further comprises a quaternary amine compound.
  • the disclosure provides a method of reducing the size of a tumor or inhibiting growth of a tumor in a subject in need thereof comprising administering any of the above polynucleotides, vector, or compositions in the subject.
  • the polynucleotide, vector, or composition is administered subcutaneously, intravenously, intraperitoneally, or intratumorally.
  • the administration treats a cancer.
  • the polynucleotide is administered intratumorally to the subject.
  • the polynucleotide is administered at an amount between about 0.10 ⁇ g per tumor and about 1000 mg per tumor.
  • the method further comprises administering an anti-cancer agent.
  • the anti-cancer agent comprises (i) an antibody or antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1 (anti-PD-1 antibody or anti-PD-Ll antibody, respectively) or a polynucleotide encoding the anti-PD-1 or anti-PD-Ll antibody or antigen-binding fragment thereof, (ii) an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4 (anti-CTLA-4 antibody) or a polynucleotide encoding the anti-CTLA-4 antibody or antigen-binding fragment thereof, or (iii) an anti-PD-1 or anti-PD-Ll antibody or antigen-binding fragment thereof or a polynucleotide encoding the anti-PD-1 or anti-PD-LI antibody or antigen- binding fragment thereof, and an anti-CTLA-4 antibody or antigen-binding fragment thereof or a polynucleot
  • the administration reduces the size of a tumor or inhibits growth of a tumor at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, or at least 5 fold better than (i) an administration of a polynucleotide encoding IL-12 alone, (ii) an administration of the anti-PD-1 or anti-PD-Ll antibody alone, or (iii) an administration of the anti-CTLA-4 antibody alone.
  • the polynucleotide encoding the anti-PD-1, anti-PD-Ll, or anti- CTLA-4 antibody or antigen-binding fragment thereof comprises an mRNA.
  • the polynucleotide encoding the anti-PD-1, anti-PD-Ll, or anti- CTLA-4 antibody or antigen-binding fragment thereof comprises at least one chemically modified nucleoside.
  • the at least one chemically modified nucleoside is selected from any chemically modified nucleoside disclosed herein and a combination thereof.
  • the at least one chemically modified nucleoside is selected from the group consisting of pseudouridine, Nl-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and a combination thereof.
  • the mRNA encoding the anti-PD-1, anti-PD-Ll, or anti-CTLA-4 antibody or antigen-binding fragment thereof comprises an open reading frame.
  • the anti-PD-Ll antibody is atezolizumab, avelumab, or durvalumab.
  • the anti-CTLA-4 antibody is tremelimumab or ipilimumab.
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL- 12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL- 12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain derived from a Type I integral membrane protein, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the transmembrane domain is selected from the group consisting of: a Cluster of Differentiation 8 (CD8) transmembrane domain, a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain, and a Cluster of Differentiation 80 (CD80)
  • CD8 Cluster of Differentiation 8
  • PDGFR Platelet-Derived Growth Factor Receptor
  • CD80 Cluster of Differentiation 80
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL- 12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a Cluster of Differentiation 8 (CD8) transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • the transmembrane domain is a CD8 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 101.
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL- 12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the transmembrane domain is a PDGFR transmembrane domain comprising a PDGFR-beta transmembrane domain.
  • the PDGFR-beta transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 102.
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL- 12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a Cluster of Differentiation 80 (CD80) transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the transmembrane domain is a CD80 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 103.
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL- 12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain and an intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the intracellular domain is derived from the same polypeptide as the transmembrane domain. In some embodiments, the intracellular domain is derived from a different polypeptide than the transmembrane domain is derived from. In some embodiments, the
  • intracellular domain is selected from the group consisting of: a PDGFR intracellular domain, a truncated PDGFR intracellular domain, and a CD80 intracellular domain.
  • the intracellular domain is a PDGFR intracellular domain comprising a PDGFR-beta intracellular domain.
  • the PDGFR-beta intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 226.
  • the truncated PDGFR intracellular domain comprises a PDGFR-beta intracellular domain truncated at E570 or G739.
  • truncated PDGFR-beta intracellular domain truncated at E570 comprises the amino acid sequence set forth in SEQ ID NO: 227.
  • the truncated PDGFR-beta transmembrane truncated at G739 comprises the amino acid sequence set forth in SEQ ID NO: 228.
  • the intracellular domain is a CD80 intracellular domain.
  • the CD80 intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 225.
  • the membrane domain comprises a PDGFR-beta
  • the membrane domain comprises a PDGFR-beta transmembrane domain and a truncated PDGFR-beta intracellular domain truncated at E570. In any of the foregoing aspects, the membrane domain comprises a PDGFR-beta transmembrane domain and a truncated PDGFR-beta intracellular domain truncated at G739. In any of the foregoing aspects, the membrane domain comprises a CD80 transmembrane domain and a CD80 intracellular domain.
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL- 12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain and a PDGFR intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • the PDGFR open reading frame
  • transmembrane domain comprises a PDGFR-beta transmembrane domain and the PDGFR intracellular domain comprises a PDGFR-beta intracellular domain.
  • the PDGFR transmembrane domain comprises a PDGFR-beta transmembrane domain and the PDGFR intracellular domain comprises a truncated PDGFR-beta intracellular domain.
  • the PDGFR-beta transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 102 and the PDGFR-beta intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 226. In some embodiments, the PDGFR-beta transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 102 and the truncated PDGFR-beta intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 227.
  • the PDGFR-beta transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 102 and the truncated PDGFR-beta intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 228.
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL- 12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a Cluster of Differentiation 80 (CD80) transmembrane domain and a CD80 intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5'
  • the CD80 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 103 and the CD80 intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 225.
  • the membrane domain is operably linked to the IL-12A polypeptide by a peptide linker. In any of the foregoing aspects, the membrane domain is operably linked to the IL-12B polypeptide by a peptide linker.
  • the membrane domain is operably linked to the IL-12A polypeptide by a Gly/Ser linker. In some aspects, the membrane domain is operably linked to the IL-12B polypeptide by a Gly/Ser linker.
  • the Gly/Ser linker comprises (G n S) m , wherein n is 1, 2 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20.
  • the Gly/Ser linker further comprises a leucine and a glutamine at the 3'end of the Gly/Ser linker. In some aspects, the linker comprises the amino acid sequence set forth in SEQ ID NO: 229.
  • the IL-12B polypeptide is operably linked to the IL-12A polypeptide by a peptide linker.
  • the IL-12B polypeptide is located at the 5' terminus of the IL-12A polypeptide, or the 5' terminus of the peptide linker.
  • the IL- 12A polypeptide is located at the 5' terminus of the IL-12B polypeptide, or the 5' terminus of the peptide linker.
  • the peptide linker comprises a Gly/Ser linker.
  • the Gly/Ser linker comprises (G n S) m , wherein n is 1, 2 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In some aspects, the Gly/Ser linker comprises (G n S) m , and wherein n is 6 and m is 1.
  • the ORF encodes a signal peptide.
  • the signal peptide is an IL-12B signal peptide.
  • the IL-12B signal peptide comprises the amino acid sequence set forth in amino acids 1 to 22 of SEQ ID NO: 48.
  • the IL-12B polypeptide comprises the amino acid sequence set forth in amino acids 23 to 328 of SEQ ID NO: 48.
  • the IL-12A polypeptide comprises the amino acid sequence set forth in amino acids 336 to 532 of SEQ ID NO: 48.
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide via a linker, wherein the membrane domain comprises a transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide via a linker, wherein the membrane domain comprises a transmembrane domain derived from a Type I integral membrane protein, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the transmembrane domain is selected from the group consisting of: a Cluster of Differentiation 8 (CD8) transmembrane domain, a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain, and a Cluster of Differentiation 80 (CD80)
  • CD8 Cluster of Differentiation 8
  • PDGFR Platelet-Derived Growth Factor Receptor
  • CD80 Cluster of Differentiation 80
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide via a linker, wherein the membrane domain comprises a Cluster of Differentiation 8 (CD8) transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • the transmembrane domain is a CD8 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 101.
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide via a linker, wherein the membrane domain comprises a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the transmembrane domain is a PDGFR transmembrane domain comprising a PDGFR-beta transmembrane domain.
  • the PDGFR-beta transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 102.
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide via a linker, wherein the membrane domain comprises a Cluster of Differentiation 80 (CD80) transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the transmembrane domain is a CD8 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 103.
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide via a linker, wherein the membrane domain comprises a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain and a PDGFR intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • the PDGFR Platelet-Derived Growth Factor Receptor
  • transmembrane domain comprises a PDGFR-beta transmembrane domain and the PDGFR intracellular domain comprises a PDGFR-beta intracellular domain.
  • the PDGFR transmembrane domain comprises a PDGFR-beta transmembrane domain and the PDGFR intracellular domain comprises a truncated PDGFR-beta intracellular domain.
  • the PDGFR-beta transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 102 and the PDGFR-beta intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 226. In some embodiments, the PDGFR-beta transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 102 and the truncated PDGFR-beta intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 227.
  • the PDGFR-beta transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 102 and the truncated PDGFR-beta intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 228.
  • the disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide via a linker, wherein the membrane domain comprises a Cluster of Differentiation 80 (CD80) transmembrane domain and a CD80 intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the CD80 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 103 and the CD80 intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 225.
  • the disclosure provides an mRNA comprising a 5' UTR, an open reading frame (ORF), and a 3' UTR, wherein the ORF comprises a nucleotide sequence encoding from 5' to 3':
  • IL-12B is a human IL-12 p40 subunit polypeptide
  • LI is a first peptide linker
  • IL-12A is a human IL-12 p35 subunit polypeptide
  • L2 is a second peptide linker
  • MD is a membrane domain comprising a transmembrane domain from a Type I integral membrane protein.
  • Type I integral membrane protein is selected from the group consisting of: a Cluster of Differentiation 8 (CD8) transmembrane domain, a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain, and a Cluster of Differentiation 80 (CD80) transmembrane domain.
  • CD8 Cluster of Differentiation 8
  • PDGFR Platelet-Derived Growth Factor Receptor
  • CD80 Cluster of Differentiation 80
  • the disclosure provides an mRNA comprising a 5' UTR, an open reading frame (ORF), and a 3' UTR, wherein the ORF comprises a nucleotide sequence encoding from 5' to 3':
  • IL-12B is a human IL-12 p40 subunit polypeptide
  • LI is a first peptide linker
  • IL-12A is a human IL-12 p35 subunit polypeptide
  • L2 is a second peptide linker
  • MD is a membrane domain comprising a transmembrane domain from a Type I integral membrane protein
  • Type I integral membrane protein is selected from the group consisting of: a Cluster of Differentiation 8 (CD8) transmembrane domain, a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain, and a Cluster of Differentiation 80 (CD80)
  • the disclosure provides an mRNA comprising a 5' UTR, an open reading frame (ORF), and a 3' UTR, wherein the ORF comprises a nucleotide sequence encoding from 5' to 3':
  • IL-12B is a human IL-12 p40 subunit polypeptide
  • LI is a first peptide linker
  • IL-12A is a human IL-12 p35 subunit polypeptide
  • L2 is a second peptide linker
  • MD is a membrane domain comprising a transmembrane domain from a Type I integral membrane protein and an intracellular domain.
  • the Type I integral membrane protein is selected from the group consisting of: a Cluster of Differentiation 8 (CD8) transmembrane domain, a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain, and a Cluster of Differentiation 80 (CD80) transmembrane domain.
  • CD8 Cluster of Differentiation 8
  • PDGFR Platelet-Derived Growth Factor Receptor
  • CD80 Cluster of Differentiation 80
  • the disclosure provides an mRNA comprising a 5' UTR, an open reading frame (ORF), and a 3' UTR, wherein the ORF comprises a nucleotide sequence encoding from 5' to 3':
  • IL-12B is a human IL-12 p40 subunit polypeptide
  • LI is a first peptide linker
  • IL-12A is a human IL-12 p35 subunit polypeptide
  • L2 is a second peptide linker
  • MD is a membrane domain comprising a transmembrane domain from a Type I integral membrane protein and an intracellular domain
  • Type I integral membrane protein is selected from the group consisting of: a Cluster of Differentiation 8 (CD8) transmembrane domain, a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain, and a Cluster of Differentiation 80 (CD80)
  • the disclosure provides an mRNA comprising a 5' UTR, an open reading frame (ORF), and a 3' UTR, wherein the ORF comprises a nucleotide sequence encoding from 5' to 3':
  • IL-12B is a human IL-12 p40 subunit polypeptide
  • LI is a first peptide linker
  • IL-12A is a human IL-12 p35 subunit polypeptide
  • L2 is a second peptide linker
  • MD is a membrane domain comprising a Cluster of Differentiation Factor 80 (CD80) transmembrane domain.
  • CD80 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 103.
  • the disclosure provides an mRNA comprising a 5' UTR, an open reading frame (ORF), and a 3' UTR, wherein the ORF comprises a nucleotide sequence encoding from 5' to 3':
  • IL-12B is a human IL-12 p40 subunit polypeptide
  • LI is a first peptide linker
  • IL-12A is a human IL-12 p35 subunit polypeptide
  • L2 is a second peptide linker
  • MD is a membrane domain comprising a Cluster of Differentiation Factor 80 (CD80) transmembrane domain and a CD80 intracellular domain.
  • CD80 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 103.
  • CD80 intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 225.
  • the MD comprises a CD80 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 103, and a CD80 intracellular domain comprising the amino acid sequence set forth in SEQ ID NO: 225.
  • the disclosure provides an mRNA comprising a 5' UTR, an open reading frame (ORF), and a 3' UTR, wherein the ORF comprises a nucleotide sequence encoding from 5' to 3':
  • IL-12B is a human IL-12 p40 subunit polypeptide
  • LI is a first peptide linker
  • IL-12A is a human IL-12 p35 subunit polypeptide
  • L2 is a second peptide linker
  • MD is a membrane domain comprising a PDGFR transmembrane domain.
  • the PDGFR transmembrane domain comprises a PDGFR-beta transmembrane domain.
  • the PDGFR-beta transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 102.
  • the disclosure provides an mRNA comprising a 5' UTR, an open reading frame (ORF), and a 3' UTR, wherein the ORF comprises a nucleotide sequence encoding from 5' to 3':
  • IL-12B is a human IL-12 p40 subunit polypeptide
  • LI is a first peptide linker
  • IL-12A is a human IL-12 p35 subunit polypeptide
  • L2 is a second peptide linker
  • MD is a membrane domain comprising a PDGFR transmembrane domain and a PDGFR intracellular domain.
  • the PDGFR transmembrane domain comprises a PDGFR- beta transmembrane domain.
  • the PDGFR-beta transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 102.
  • the PDGFR intracellular domain comprises a PDGFR-beta intracellular domain.
  • the PDGFR-beta intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 226.
  • the PDGFR intracellular domain comprises a truncated PDGFR-beta intracellular domain.
  • the truncated PDGFR-beta intracellular domain is truncated at E570 and comprises the amino acid sequence set forth in SEQ ID NO: 227. In some aspects, the truncated PDGFR-beta intracellular domain is truncated at G739 and comprises the amino acid sequence set forth in SEQ ID NO: 228. In some aspects, the MD comprises a PDGFR-beta transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 102, and a PDGFR-beta intracellular domain comprising the amino acid sequence set forth in SEQ ID NO: 226.
  • the MD comprises a PDGFR-beta transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 102, and a truncated PDGFR-beta intracellular domain comprising the amino acid sequence set forth in SEQ ID NO: 227.
  • the MD comprises a PDGFR-beta transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 102, and a truncated PDGFR-beta intracellular domain comprising the amino acid sequence set forth in SEQ ID NO: 228.
  • the ORF of the mRNA encodes a signal peptide.
  • the signal peptide is an IL-12B signal peptide.
  • the IL-12B signal peptide comprises the amino acid sequence set forth in amino acids 1 to 22 of SEQ ID NO: 48.
  • the ORF of the mRNA encodes an IL-12B polypeptide comprising the amino acid sequence set forth in amino acids 23 to 328 of SEQ ID NO: 48.
  • the ORF of the mRNA encodes an IL-12B polypeptide comprising the amino acid sequence set forth in amino acids 336 to 532 of SEQ ID NO: 48.
  • first peptide linker [LI] and second peptide linker [L2] are each a Gly/Ser linker.
  • [LI] comprises SEQ ID NO: 214.
  • [L2] comprises (G n S) m , wherein n is 1-4, 1, 2, 3 or 4 and m is 1-4, 1, 2, 3, or 4.
  • [L2] comprises (G 4 S) m , wherein m is 1-4, 1, 2, 3, or 4.
  • [L2] comprises the amino acid sequence set forth in SEQ ID NO: 229.
  • the ORF of the mRNA comprises the sequence set forth in SEQ ID NO: 273 or SEQ ID NO: 274, or a nucleotide sequence at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 273 or SEQ ID NO: 274.
  • the ORF of the mRNA comprises the sequence set forth in any one of SEQ ID NOs: 275-279, or a nucleotide sequence at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in any one of SEQ ID NOs: 275-279.
  • the ORF of the mRNA comprises the sequence set forth in SEQ ID NO: 281 , or a nucleotide sequence at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 281.
  • the ORF of the mRNA comprises the sequence set forth in SEQ ID NO: 282 , or a nucleotide sequence at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to a sequence set forth in SEQ ID NO: 282.
  • the 3'UTR of the polynucleotide or mRNA comprises a microRNA binding site.
  • the microRNA binding site is a miR-122 binding site.
  • the miR-122 binding site is a miR-122-3p or miR-122-5p binding site.
  • the miR-122-5p binding site comprises the sequence set forth in SEQ ID NO: 54.
  • the 3'UTR comprises a sequence set forth in SEQ ID NO: 283.
  • the 5'UTR of the polynucleotide or mRNA comprises a sequence set forth in SEQ ID NO: 287.
  • the polynucleotide or mRNA comprises a 5 'terminal cap structure.
  • the 5' terminal cap structure is a CapO, Capl, ARCA, inosine, Nl- methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5'methylGcap, or an analog thereof.
  • the polynucleotide or mRNA comprises a 3' polyA tail.
  • the polynucleotide or mRNA comprises at least one chemical modification.
  • the chemical modification is selected from the group consisting of pseudouridine, Nl-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l- methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5- aza- uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-0- methyl
  • the chemical modification is selected from the group consisting of pseudouridine or a pseudouridine analog. In some aspects, the chemical modification is Nl- methylpseudouridine. In some aspects, the mRNA is fully modified with Nl-methylpseudouridine.
  • the disclosure provides a composition comprising a polynucleotide or mRNA as described herein, and a pharmaceutically acceptable carrier.
  • the disclosure provides a lipid nanoparticle comprising a polynucleotide or mRNA as described herein.
  • the disclosure provides a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12
  • ORF open reading frame
  • IL-12 human interleukin-12
  • polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • IL-12B IL-12 p40 subunit
  • IL-12A IL-12A
  • the membrane domain comprises a transmembrane domain
  • polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • the disclosure provides a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL- 12A) polypeptide, wherein the membrane domain comprises a transmembrane domain and an intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL- 12A) polypeptide, wherein the membrane domain comprises a Cluster of Differentiation 8 (CD8) transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL- 12A) polypeptide, wherein the membrane domain comprises a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL- 12A) polypeptide, wherein the membrane domain comprises a Cluster of Differentiation 80 (CD80) transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12
  • ORF open reading frame
  • IL-12 human interleukin-12
  • polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain and a PDGFR intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • IL-12B IL-12 p40 subunit
  • IL-12A IL-12A
  • the membrane domain comprises a Platelet-Derived Growth Factor Receptor (PDGFR) transmembrane domain and a PDGFR intracellular domain
  • PDGFR Platelet-Derived Growth Factor Receptor
  • the disclosure provides a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a Cluster of Differentiation 80 (CD80) transmembrane domain and a CD80 intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the lipid nanoparticle comprises a molar ratio of about 20-60% ionizable amino lipid: 5-25% phospholipid: 25-55% sterol; and 0.5-15% PEG-modified lipid.
  • the ionizable amino lipid is selected from the group consisting of for example, 2,2- dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4-
  • the ionizable amino lipid comprises a compound of Formula (I).
  • the compound of Formula (I) is Compound 18.
  • the disclosure provides a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a Cluster of Differentiation 80 (CD80) transmembrane domain and a CD80 intracellular domain, wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR, and wherein the lipid nanoparticle comprising a molar raiot of about 20-60% ionizable amino lipid: 5-25% phospholipid: 25-55% sterol; and 0.5-15% PEG-modified lipid.
  • the disclosure provides a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a Cluster of Differentiation 80 (CD80) transmembrane domain and a CD80 intracellular domain, wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR, wherein the lipid nanoparticle comprising a molar raiot of about 20-60% ionizable amino lipid: 5-25% phospholipid: 25-55% sterol; and 0.5-15% PEG-modified lipid, and
  • the disclosure provides a pharmaceutical composition comprising a lipid nanoparticle as described herein, and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier comprises a buffer solution.
  • the pharmaceutically acceptable carrier comprises a buffer solution.
  • composition is formulated for intratumoral delivery.
  • the disclosure provides a polynucleotide, mRNA, composition, lipid nanoparticle, or pharmaceutical composition as described herein, for use in treating or delaying progression of cancer in an individual, wherein the treatment comprises administration of the polynucleotide, mRNA, composition, lipid nanoparticle or pharmaceutical composition in combination with a second composition, wherein the second composition comprises anti-cancer agent, or a polynucleotide comprising an ORF encoding an anti-cancer agent, such as a checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier.
  • the disclosure provides a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR, for use in treating or delaying progression of cancer in an individual, wherein the treatment comprises administration of the lipid nanoparticle in combination with a second composition, wherein the second composition comprises anti-cancer agent, or a polynucleotide comprising an ORF encoding an anti-cancer agent, such as a checkpoint
  • the disclosure provides use of a polynucleotide, mRNA, composition, lipid nanoparticle, or pharmaceutical composition described herein in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament comprises the polynucleotide, mRNA, composition, lipid nanoparticle, or pharmaceutical composition, and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administration of the medicament in combination with a composition comprising an anti-cancer agent, or a polynucleotide comprising an ORF encoding an anti-cancer agent, such as a checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier.
  • the disclosure provides use of a a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL- 12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR, in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament comprises the lipid nanoparticle and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administration of the medicament in combination with a composition comprising an anti-cancer agent, or a polynucleotide
  • the disclosure provides a kit comprising a container comprising a polynucleotide, mRNA, composition, the lipid nanoparticle, or pharmaceutical composition as described herein, and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the polynucleotide, mRNA, composition, lipid nanoparticle or pharmaceutical composition, for treating or delaying progression of cancer in an individual.
  • the package insert further comprises instructions for administration of the lipid nanoparticle or pharmaceutical composition in combination with a composition comprising an anti-cancer agent, or a polynucleotide comprising an ORF encoding an anti-cancer agent, such as a checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier, for treating or delaying progression of cancer in an individual.
  • a composition comprising an anti-cancer agent, or a polynucleotide comprising an ORF encoding an anti-cancer agent, such as a checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier, for treating or delaying progression of cancer in an individual.
  • the disclosure provides a kit comprising a container comprising a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL- 12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR, and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the lipid nanoparticle or treating or delaying progression of cancer in an individual.
  • ORF open reading frame
  • the disclosure provides a kit comprising a polynucleotide, mRNA, composition, the lipid nanoparticle, or pharmaceutical composition as described herein, and a package insert comprising instructions for administration of the medicament alone, or in
  • kits further comprises a package insert comprising instructions for administration of the first medicament prior to, current with, or subsequent to administration of the second medicament for treating or delaying progression of cancer in an individual.
  • the disclosure provides a kit comprising a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL- 12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR, and a package insert comprising instructions for administration of the medicament alone, or in combination with a composition comprising an anti-cancer agent, or a polynucleotide comprising an ORF encoding an anti-cancer agent, such as a checkpoint inhibitor polypeptide, and an optional pharmaceutically
  • the checkpoint inhibitor polypeptide inhibits PD1, PD-L1, CTLA4, or a combination thereof.
  • the checkpoint inhibitor polypeptide is an antibody.
  • the checkpoint inhibitor polypeptide is an antibody selected from an anti- CTLA4 antibody or antigen-binding fragment thereof that specifically binds CTLA4, an anti-PDl antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-Ll antibody or antigen -binding fragment thereof that specifically binds PD-L1, and a combination thereof.
  • the checkpoint inhibitor polypeptide is an anti-PD-Ll antibody selected from atezolizumab, avelumab, or durvalumab.
  • the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody selected from tremelimumab or ipilimumab. In some aspects, the checkpoint inhibitor polypeptide is an anti-PDl antibody selected from nivolumab or pembrolizumab.
  • the disclosure provides a method of reducing the size of a tumor or inhibiting growth of a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a polynucleotide, mRNA, composition, lipid nanoparticle or pharmaceutical composition as described herein.
  • the disclosure provides a method of reducing the size of a tumor or inhibiting growth of a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a method of reducing the size of a tumor or inhibiting growth of a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain and an intracellular domain, and wherein the
  • polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • UTR 5' untranslated region
  • the disclosure provides a method of reducing the size of a tumor or inhibiting growth of a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a CD80 transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a method of reducing the size of a tumor or inhibiting growth of a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a PDGFR transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a method of reducing the size of a tumor or inhibiting growth of a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a CD8 transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a method of reducing the size of a tumor or inhibiting growth of a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a PDGFR transmembrane domain and a PDGFR intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a method of reducing the size of a tumor or inhibiting growth of a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a CD80 transmembrane domain and a CD80 intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a method of inducing an anti-tumor response in a subject in need thereof, comprising administering to the subject an effective amount of a
  • polynucleotide polynucleotide, mRNA, composition, lipid nanoparticle or pharmaceutical composition as described herein.
  • the disclosure provides a method of inducing an anti-tumor response in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL- 12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a method of inducing an anti-tumor response in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL- 12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain and an intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a method of inducing an anti-tumor response in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL- 12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a PDGFR transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a method of inducing an anti-tumor response in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL- 12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a CD80 transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a method of inducing an anti-tumor response in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL- 12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a CD8 transmembrane domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a method of inducing an anti-tumor response in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL- 12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a CD80 transmembrane domain and a CD80 intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the disclosure provides a method of inducing an anti-tumor response in a subject in need thereof, comprising administering to the subject an effective amount of a lipid nanoparticle comprising a polynucleotide comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL- 12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a PDGFR transmembrane domain and a PDGFR intracellular domain, and wherein the polynucleotide is an mRNA comprising a 5' untranslated region (UTR), the ORF, and a 3' UTR.
  • ORF open reading frame
  • the polynucleotide, mRNA, composition, lipid nanoparticle lipid or pharmaceutical composition is administered by intratumoral injection.
  • the anti-tumor response comprises a T-cell response.
  • the T-cell response comprises CD8+ T cells.
  • the method further comprises administering to the subject an effective amount of a composition comprising an anti-cancer agent, or a polynucleotide comprising an ORF encoding an anti-cancer agent.
  • the method further comprises administering a second composition comprising a checkpoint inhibitor polypeptide or polynucleotide encoding the same, and an optional pharmaceutically acceptable carrier.
  • the checkpoint inhibitor polypeptide inhibits PD1, PD-L1, CTLA4, or a combination thereof.
  • the checkpoint inhibitor polypeptide is an antibody.
  • the checkpoint inhibitor polypeptide is an antibody selected from an anti-CTLA4 antibody or antigen-binding fragment thereof that specifically binds CTLA4, an anti-PDl antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-Ll antibody or antigen-binding fragment thereof that specifically binds PD-L1, and a combination thereof.
  • the checkpoint inhibitor polypeptide is an anti-PD-Ll antibody selected from atezolizumab, avelumab, or durvalumab. In some aspects, the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody selected from tremelimumab or ipilimumab. In some aspects, the checkpoint inhibitor polypeptide is an anti-PDl antibody selected from nivolumab or pembrolizumab.
  • composition comprising the checkpoint inhibitor polypeptide is administered by intravenous injection. In some aspects, the composition comprising the checkpoint inhibitor polypeptide is administered once every 2 to 3 weeks.
  • the second composition comprising the checkpoint inhibitor polypeptide is administered prior to, concurrent with, or subsequent to administration of the polynucleotide, mRNA, composition, lipid nanoparticle or pharmaceutical composition.
  • FIGs. 1A-1D show exemplary structures of tethered IL-12 polypeptides with (FIGs. 1A and 1C) or without (FIGs. IB and ID) a linker between an IL-12 polypeptide ("IL12") and a membrane domain (“MD").
  • the "IL12" polypeptide includes polypeptides comprising IL-12A, IL-12B, or both IL-12A and IL-12B.
  • “N” indicates the amino-terminus of the polypeptide
  • C indicates the carboxy-terminus of the polypeptide.
  • FIGs. 2A-2E show structures of tethered murine IL-12 polypeptides, comprising an IL-12B subunit linked to an IL-12A subunit via a linker, as used in the Examples.
  • FIG. 2A shows an IL-12 polypeptide linked to a CD8 transmembrane domain via a linker, with a V5 tag ("mIL12-8TM").
  • FIG. 2B shows an IL-12 polypeptide linked to a PGFRB transmembrane domain via a linker, with a V5 tag ("mIL12-PTM").
  • FIG. 2C shows an IL-12 polypeptide linked to a CD80 transmembrane domain and intracellular domain via a linker ("mIL12-80TID").
  • FIG. 2D shows an IL-12 polypeptide linked to a CD80 transmembrane domain, with no linker, and a V5 tag ("mIL12- 80TM”).
  • FIG. 2E shows an IL-12 polypeptide linked to a CD80 transmembrane and intracellular domain without a linker, and comprising a hemagglutinin (HA) tag and IgK signal peptide ("IgK_mscIL12-80TID").
  • HA hemagglutinin
  • FIG. 3 is a graph depicting in vitro expression levels of IL-12 in the supernatant or lysates of HeLa cells after 24 hours of exposure to transfection reagent with no mRNA ("Mock") or transfected in individual cell culture wells with the following constructs: mRNA encoding a secreted mouse IL- 12 polypeptide ("mIL12AB”), mIL12-8TM, mIL12-PTM, and mIL12-80TM. The amount of IL-12 in nanograms per respective culture well (“ng/well”) is shown on the y-axis of the figure.
  • FIGs. 4A-4D show the in vitro bioactivity associated with various tethered IL-12 polypeptide constructs.
  • FIGs. 4A and 4C show the level of proliferation of mouse splenic CD8+ T cell in relative light units ("RLU") on the y-axis.
  • FIGs. 4B and 4D show the amount of interferon gamma (IFNy) secretion by mouse splenic CD8+ T cells in nanograms per milliliter (“ng/ml”) on the y-axis. Proliferation of and secretion by CD8+ T cells in each of FIGs.
  • IFNy interferon gamma
  • FIGs. 4A-4D were measured after 72 hours in co-culture with "Mock” transfected HeLa cells or HeLa cells transfected in individual culture wells with mIL12AB, mIL12-8TM, or mIL12-80TM as described in the brief description of FIG. 3.
  • Recombinant mouse IL-12 (rmIL12) was also added to a subset of "Mock” cultures (“Mock + rmIL12").
  • Each condition in FIGs. 4A-4D represent 50,000 CD8+ T cells cultured with a fixed number of HeLa cells from a HeLa cell culture transfected with one of the noted constructs and further including a fixed amount of supernatant from the same HeLa cell culture.
  • FIGs. 5A and 5B show in vitro IL-12 protein expression and bioactivity on mouse splenic CD8+ T cells of HeLa cells transfected with IL-12 polypeptide constructs.
  • FIG. 5A shows the amount of IL-12 (ng/well) in the supernatant or lysates of "Mock” HeLa cells or HeLa cells 24 hours after transfection in individual cell culture wells with various tethered murine IL-12 mRNAs.
  • FIG. 5B shows the amount of IFNy secretion (ng/mL) by mouse splenic CD8+ T cells after 72 hours of co-culture with "Mock” or transfected HeLa cells and supernatant.
  • FIGs. 6A and 6B show the plasma levels of IL-12 (FIG. 6A) and IFNy (FIG. 6B) from mice having MC38 tumors, after treatment with mIL12AB ("secreted mIL-12") or mIL12-PTM ("tethered mIL-12"). Each graph shows plasma concentration in picograms per milliliter (“pg/mL”) over time post dose in hours.
  • FIG. 7 provides graphs showing the percentage of body weight change over time (days) post implant of MC38 tumors into mice.
  • the vertical line indicates the day treatment began with either negative control (NST mRNA; left), secreted IL- 12 (mIL12AB; middle) or tethered IL-12 (mIL12- PTM; right).
  • NST mRNA negative control
  • mIL12AB secreted IL- 12
  • mIL12- PTM tethered IL-12
  • FIGs. 8A-8E show in vivo tumor efficacy in both primary treated and secondary untreated (i.e., distal) tumors with either a negative control mRNA encoding an untranslatable sequence of mOX40L ("Negative Control"), or mIL12-PTM mRNA construct as described in the brief description of FIG. 2B.
  • FIG. 8A shows a schematic description of the MC38 dual flank model used in the experiments. "5e5" indicates that 5 x 10 5 MC38 cells were inoculated into the primary (right) or secondary (left) flanks to produce the tumors.
  • FIG. 8B-8E show tumor volume in cubic millimeters (mm 3 ) at the number of days indicated on the x-axis post implant with MC38.
  • FIG. 8B shows the effect of the negative control mRNA on the primary treated tumor.
  • FIG. 8C shows the effect of the negative control mRNA on the secondary tumor.
  • FIG. 8D shows the effect of mIL12-PTM on the primary treated tumor.
  • FIG. 8E shows the effect of mIL12-PTM on the secondary tumor.
  • FIGs. 9A-9D show structures of tethered human IL-12 polypeptides, comprising an IL- 12B subunit linked to an IL-12A subunit via a linker, as used in the Examples.
  • FIG. 9A shows an IL-12 polypeptide linked to a CD8 transmembrane domain via a linker, with a V5 tag ("ML12-8TM").
  • FIG. 9B shows an IL-12 polypeptide linked to a CD80 transmembrane domain and intracellular domain via a linker ("hIL12-80TID").
  • FIG. 9C shows an IL-12 polypeptide linked to a PGFRB transmembrane domain and truncated intracellular domain (E570tr), via a linker ("ML12- PTIDE570").
  • FIG. 9D shows an IL-12 polypeptide linked to a PGFRB transmembrane domain and truncated intracellular domain (G739tr), via a linker ("ML12-PTIDG739").
  • FIGs. 10A and 10B show in vitro IL-12 protein expression and bioactivity on human peripheral blood CD8+ T cells of HeLa cells transfected with IL-12 polypeptide constructs.
  • FIG. 10A shows the amount of IL-12 (ng/well) in the supernatant or lysates of "Mock” HeLa cells or HeLa cells 24 hours after transfection in individual cell culture wells with various tethered human IL-12 mRNAs 24 hours after transfection.
  • FIG. 10B shows the amount of IFNy secretion (ng/mL) by human peripheral blood CD8+ T cells after 72 hours of co-culture with "Mock” or transfected HeLa cells and supernatants.
  • FIG. 11 shows in vitro IL-12 protein expression of ML12-80TID encoded by four different mRNA sequences.
  • the graph shows the amount of IL-12 (ng/well) in the supernatant (left) or lysates (right) of "Mock" HeLa cells or HeLa cells transfected in individual wells with the various mRNAs 24 hours after transfection.
  • the present disclosure provides a new approach to treat cancer involving the prevention or treatment of disease with substances (e.g., mRNAs encoding a tethered IL-12 polypeptide, which comprises an IL-12 polypeptide and a membrane domain as disclosed herein) that stimulate the immune response, i.e., immunotherapy.
  • substances e.g., mRNAs encoding a tethered IL-12 polypeptide, which comprises an IL-12 polypeptide and a membrane domain as disclosed herein
  • the disclosure relates to methods of treating cancer using a polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide.
  • An IL-12 polypeptide as disclosed herein comprises IL-12A, IL-12B, or both IL-12A and IL-12B.
  • the disclosure provides methods of treating cancer using a combination approach that features a polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide and an anti-cancer agent, e.g., an immune-checkpoint inhibitor, e.g., anti-PD-1 antibody, anti-PD-Ll antibody, and/or anti-CTLA-4 antibody.
  • an immune-checkpoint inhibitor e.g., anti-PD-1 antibody, anti-PD-Ll antibody, and/or anti-CTLA-4 antibody.
  • priming of an anti-cancer immune response is possible by administering, e.g. , intratumorally, a polynucleotide (e.g., mRNA) encoding a tethered IL- 12 polypeptide in the stimulation of, for example, T-cells and/or natural killer cells. Therefore, a polynucleotide (e.g., mRNA) encoding a tethered IL- 12 polypeptide is believed to provide a first stimulation signal to the immune system, for example, within the tumor environment, e.g. , via intratumoral injection of the polynucleotide (e.g., mRNA).
  • a polynucleotide e.g., mRNA
  • IL- 12 can also stimulate the production of interferon-gamma (IFN- ⁇ ) and tumor necrosis factor-alpha (TNF-a) from T cells and natural killer (NK) cells.
  • IFN- ⁇ interferon-gamma
  • TNF-a tumor necrosis factor-alpha
  • IL- 12 either directly or indirectly through IFN- ⁇ , can also increase expression of PD-L1 in tumor cells, which can impair local tumor immunity. Therefore, in some aspects, the disclosure provides a method of treating a tumor comprising administering a
  • the disclosure includes a method of treating a tumor comprising administering a polynucleotide (e.g., mRNA) encoding a tethered IL- 12 polypeptide in combination with an anti-CTLA-4 antibody.
  • a polynucleotide e.g., mRNA
  • the disclosure provides a method of treating a tumor comprising administering a polynucleotide (e.g., mRNA) encoding a tethered IL- 12 polypeptide in combination with an anti-PD- 1 antibody or anti-PD-Ll antibody and an anti-CTLA-4 antibody.
  • a polynucleotide e.g., mRNA
  • anti-PD- 1 antibody or anti-PD-Ll antibody can be administered in the form of a polynucleotide.
  • the anti-CTLA-4 antibody can be administered in the form of a polynucleotide.
  • Exemplary aspects feature treatment with lipid nanoparticle- (LNP-) encapsulated mRNAs.
  • LNP- lipid nanoparticle-
  • Exemplary aspects feature intratumoral administration of mRNAs in cationic lipid-based LNPs.
  • Certain aspects of the present disclosure are directed to methods of reducing or decreasing size, mass, and/or volume of a tumor or preventing the growth of a tumor in a subject in need thereof comprising administering a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide disclosed herein, or a vector or a host cell comprising the polynucleotide, or a tethered IL- 12 polypeptide encoded by the polynucleotide.
  • a polynucleotide e.g., mRNA, encoding a tethered IL-12 polypeptide disclosed herein
  • a vector or a host cell comprising the polynucleotide, or a tethered IL- 12 polypeptide encoded by the polynucleotide.
  • the present disclosure provides methods of promoting an anti-tumor effect (e.g., induce T cell proliferation, induce T cell infiltration in a tumor, induce a memory T cell response, increasing the number of NK cells, etc.) by administering the polynucleotide (e.g., mRNA) encoding a tethered IL- 12 polypeptide or the polynucleotide in combination with any agents disclosed herein.
  • an anti-tumor effect e.g., induce T cell proliferation, induce T cell infiltration in a tumor, induce a memory T cell response, increasing the number of NK cells, etc.
  • the present disclosure provides a method of activating T cells in a subject in need thereof, inducing T cell proliferation in a subject in need thereof, inducing T cell infiltration in a tumor of a subject in need thereof, and/or inducing a memory T cell response in a subject in need thereof, comprising administering to the subject a polynucleotide encoding a tethered IL- 12 polypeptide alone or in combination with a second agent, e.g., a checkpoint inhibitor, e.g., an anti-PD- 1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody.
  • a second agent e.g., a checkpoint inhibitor, e.g., an anti-PD- 1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody.
  • the intratumoral administration of the polynucleotide e.g., mRNA
  • a tethered IL- 12 polypeptide alone or in combination with a second agent
  • the intratumoral administration of the polynucleotide can increase the efficacy of the anti-tumor effect (e.g. , T cell infiltration in a tumor) compared to other routes of administration.
  • cytokines such as IL- 12
  • IL- 12 has been associated with toxicities in treated subjects. See, e.g. , Leonard, J. P. et ah, Blood 90: 2541-2548 (1997).
  • the present disclosure provides a method of reducing the size of a tumor or inhibiting the growth of a tumor in a subject in need thereof with reduced toxicity, comprising administering to the subject a polynucleotide (e.g., mRNA) encoding a tethered IL- 12 polypeptide disclosed herein.
  • a polynucleotide e.g., mRNA
  • the administration exhibits reduced toxicity compared to an administration of a reference polynucleotide (e.g., mRNA) encoding an IL- 12 polypeptide that is not tethered.
  • the reduced toxicity is a reduction in a toxicity or a toxic effect selected from the group consisting of: systemic toxicity, sepsis-like syndrome, septic shock, cachexia, loss of weight, muscle atrophy, fatigue, weakness, significant loss of appetite, hepatotoxicity, a decrease in circulating leukocytes, thrombocytopenia, anemia, dyspnea, stomatitis, leukopenia,
  • hyperbilirubinemia elevations in transaminases, thrombocytopenia, organ failure, respiratory failure, liver failure, renal failure, gastrointestinal bleeding, and combinations thereof.
  • activated T cells in the subject reduce the size of a tumor or inhibit the growth of a tumor in the subject.
  • Activation of T cells can be measured using applications in the art such as measuring T cell proliferation; measuring cytokine production with enzyme-linked immunosorbent assays (ELISA) or enzyme-linked immunospot assays (ELISPOT); or detection of cell-surface markers associated with T cell activation (e.g., CD69, CD40L, CD137, CD25, CD71, CD26, CD27, CD28, CD30, CD154, and CD134) with techniques such as flow cytometry.
  • T cell proliferation in the subject is directed to an anti-tumor immune response in the subject.
  • the T cell proliferation in the subject reduces or decreases the size of a tumor or inhibits the growth of a tumor in the subject.
  • T cell proliferation can be measured using applications in the art such as cell counting, viability staining, optical density assays, or detection of cell-surface markers associated with T cell activation (e.g., CD69, CD40L, CD137, CD25, CD71, CD26, CD27, CD28, CD30, CD154, and CD134) with techniques such as flow cytometry.
  • T cell infiltration in a tumor of the subject is directed to an anti-tumor immune response in the subject.
  • the T cell infiltration in a tumor of the subject reduces or decreases the size of a tumor or inhibits the growth of a tumor in the subject.
  • T cell infiltration in a tumor can be measured using applications in the art such as tissue sectioning and staining for cell markers, measuring local cytokine production at the tumor site, or detection of T cell-surface markers with techniques such as flow cytometry.
  • the memory T cell response in the subject is directed to an anti-tumor immune response in the subject.
  • the memory T cell response in the subject reduces or decreases the size of a tumor or inhibits the growth of a tumor in the subject.
  • a memory T cell response can be measured using applications in the art such as measuring T cell markers associated with memory T cells, measuring local cytokine production related to memory immune response, or detecting memory T cell-surface markers with techniques such as flow cytometry.
  • the T cells activated by the present methods are CD4 + cells, CD8 + cells, CD62 + (L-selectin + ) cells, CD69 + cells, CD40L + cells, CD137 + cells, CD25 + cells, CD71 + cells, CD26 + cells, CD27 + cells, CD28 + cells, CD30 + cells, CD45 + cells, CD45RA + cells, CD45RO + cells, CDl lb + cells, CD154 + cells, CD134 + cells, CXCR3 + cells, CCR4 + cells, CCR6 + cells, CCR7 + cells, CXCR5 + cells, Crth2 + cells, gamma delta T cells, or any combination thereof.
  • the T cells activated by the present methods are Thi cells.
  • the T cells activated by the present methods are Th 2 cells.
  • the T cells activated by the present methods are cytotoxic T cells.
  • the infiltrating T cells induced by the present methods are CD4 + cells, CD8 + cells, CD62 + (L-selectin + ) cells, CD69 + cells, CD40L + cells, CD137 + cells, CD25 + cells, CD71 + cells, CD26 + cells, CD27 + cells, CD28 + cells, CD30 + cells, CD45 + cells, CD45RA + cells, CD45RO + cells, CDl lb + cells, CD154 + cells, CD134 + cells, CXCR3 + cells, CCR4 + cells, CCR6 + cells, CCR7 + cells, CXCR5 + cells, Crth2 + cells, gamma delta T cells, or any combination thereof.
  • the infiltrating T cells induced by the present methods are Thi cells. In other embodiments, the infiltrating T cells induced by the present methods are Th 2 cells. In other embodiments, the infiltrating T cells induced by the present methods are cytotoxic T cells.
  • the memory T cells induced by the present methods are CD4 + cells, CD8 + cells, CD62 + (L-selectin + ) cells, CD69 + cells, CD40L + cells, CD137 + cells, CD25 + cells, CD71 + cells, CD26 + cells, CD27 + cells, CD28 + cells, CD30 + cells, CD45 + cells, CD45RA + cells, CD45RO + cells, CDl lb + cells, CD154 + cells, CD134 + cells, CXCR3 + cells, CCR4 + cells, CCR6 + cells, CCR7 + cells, CXCR5 + cells, Crth2 + cells, gamma delta T cells, or any combination thereof.
  • the memory T cells induced by the present methods are Thi cells.
  • the memory T cells induced by the present methods are Th 2 cells.
  • the memory T cells induced by the present methods are cytotoxic T cells.
  • the disclosure provides a method of inducing an adaptive immune response, an innate immune response, or both adaptive and innate immune response against a tumor, comprising administering a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide alone or in combination with a second agent, e.g., a checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody, and/or any other agents disclosed herein.
  • a polynucleotide e.g., mRNA
  • a checkpoint inhibitor e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody, and/or any other agents disclosed herein.
  • the disclosure provides a method of inducing an adaptive immune response, an innate immune response, or both adaptive and innate immune response against a tumor, comprising administering a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide in combination with a second agent, e.g., a checkpoint inhibitor, e.g., an anti-PD- 1 antibody, an anti- PD-Ll antibody, and/or an anti-CTLA-4 antibody, and/or any other agents disclosed herein.
  • a polynucleotide e.g., mRNA
  • a second agent e.g., a checkpoint inhibitor, e.g., an anti-PD- 1 antibody, an anti- PD-Ll antibody, and/or an anti-CTLA-4 antibody, and/or any other agents disclosed herein.
  • the checkpoint inhibitor can be a polynucleotide (e.g., mRNA) encoding an antibody or an antigen-binding portion thereof, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody.
  • the disclosure provides a method of inducing an adaptive immune response, an innate immune response, or both adaptive and innate immune response against a tumor, comprising administering a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide.
  • the present disclosure further provides a method of increasing the number of Natural Killer (NK) cells in a subject in need thereof, comprising administering a polynucleotide comprising an mRNA encoding a tethered IL-12 polypeptide alone or in combination with a second agent, e.g., a checkpoint inhibitor, e.g., an anti-PD- 1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody, and/or any other agents disclosed herein.
  • a checkpoint inhibitor e.g., an anti-PD- 1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody, and/or any other agents disclosed herein.
  • the disclosure provides a method of increasing the number of Natural Killer (NK) cells in a subject in need thereof, comprising administering a polynucleotide comprising an mRNA encoding a tethered IL-12 polypeptide.
  • NK Natural Killer
  • the disclosure provides a method of increasing the number of Natural Killer (NK) cells in a subject in need thereof, comprising administering a polynucleotide comprising an mRNA encoding a tethered IL-12 polypeptide in combination with a second agent, e.g., a checkpoint inhibitor, e.g., an anti-PD- 1 antibody, an anti-PD-Ll antibody, and/or an anti- CTLA-4 antibody, and/or any other agents disclosed herein.
  • a checkpoint inhibitor e.g., an anti-PD- 1 antibody, an anti-PD-Ll antibody, and/or an anti- CTLA-4 antibody, and/or any other agents disclosed herein.
  • the increase in the number of NK cells in the subject is directed to an anti-tumor immune response in the subject.
  • the increase in the number of NK cells in the subject reduces or decreases the size of a tumor or inhibits the growth of a tumor in the subject.
  • Increases in the number of NK cells in a subject can be measured using applications in the art such as detection of NK cell-surface markers (e.g., CD335/NKp46; CD336/NKp44; CD337/NPp30) or intracellular NK cell markers (e.g., perforin; granzymes; granulysin).
  • NK cell-surface markers e.g., CD335/NKp46; CD336/NKp44; CD337/NPp30
  • intracellular NK cell markers e.g., perforin; granzymes; granulysin.
  • the present disclosure is also directed to a method of increasing IFNy expression in a subject having tumor comprising administering a polynucleotide encoding a tethered IL- 12 polypeptide alone or in combination with a second agent, e.g., a checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody, and/or any other agents disclosed herein.
  • the disclosure provides a method of increasing IFNy expression in a subject having tumor comprising administering a polynucleotide encoding a tethered IL- 12 polypeptide.
  • the disclosure provides a method of increasing IFNy expression in a subject having tumor comprising administering a polynucleotide encoding a tethered IL- 12 polypeptide in combination with a second agent, e.g., a checkpoint inhibitor, e.g., an anti-PD- 1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody, and/or any other agents disclosed herein.
  • a checkpoint inhibitor e.g., an anti-PD- 1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody, and/or any other agents disclosed herein.
  • inventions also include a method of increasing expression of IFNy, TNFa, IL-10, IL- 13, IL- 15/15R, IL-27, ⁇ - ⁇ , MIP- la, MCP- 1, MCP-3, M-CSF, IL-4, IL-5, or any combination thereof in a subject having tumor comprising administering a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide alone or in combination with another agent disclosed herein.
  • the methods of the present disclosure can include methods of inducing expression of GM-CSF, IL- 18, IL-3, RANTES, IL-6, or any combination thereof.
  • polynucleotide encoding a tethered IL-12 polypeptide can be formulated as a
  • tumor is used herein in a broad sense and refers to any abnormal new growth of tissue that possesses no physiological function and arises from uncontrolled usually rapid cellular proliferation.
  • tumor as used herein relates to both benign tumors and to malignant tumors.
  • Certain aspects of the disclosure provide methods of intratumorally administering a single dose of a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide alone or in
  • the disclosure provides methods of intratumorally administering a single dose of a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide. In some embodiments, the disclosure provides methods of
  • a polynucleotide e.g., mRNA
  • an mRNA encoding a tethered IL-12 polypeptide can be administered only once while the other agent can be administered regularly, following its regular dosing schedule.
  • a checkpoint inhibitor e.g., an anti-PD- 1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody
  • a polynucleotide e.g., mRNA
  • the polynucleotide is formulated in a lipid nanoparticle, e.g., Compound 18 based lipid nanoparticle, disclosed herein.
  • the intratumoral delivery of a polynucleotide encoding a tethered IL- 12 polypeptide and/or the lipid nanoparticle formulation disclosed herein allows single dose
  • this single dosing regimen of the disclosed polynucleotide can be beneficial to the subjects in need of the treatment.
  • the method comprises administering a single dose of a
  • polynucleotide e.g., mRNA
  • a second agent e.g., an anti-PD- 1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody, which can be given also in single administration or multiple administrations following its regular (e.g., approved) schedule.
  • the method comprises not more than two administrations of a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide, not more than three administrations of a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide, not more than four administrations of a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide , or not more than five administrations of a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide , optionally in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody.
  • a checkpoint inhibitor e.g., an anti-PD-1 antibody, an anti-PD-Ll
  • the present methods can result in abscopal effects, e.g., a treatment of tumor where localized treatment of a tumor, e.g., intratumoral delivery, by a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide causes not only a shrinking of the treated tumor, but also a shrinking of tumors outside the scope of the localized treatment ("distal tumor").
  • a polynucleotide e.g., mRNA
  • the administering of a polynucleotide encoding a tethered IL-12 polypeptide increases an effector to suppressor T cell ratio in the tumor.
  • the effector to suppressor T cell ratio is characterized by the ratio of (i) CD8+, CD4+, or CD8+/CD4+ T cells to (ii) Treg cells in a subject.
  • the increase in the effector to suppressor T cell ratio correlates with an increase in the number of CD8+ T cells.
  • the increase in the effector to suppressor T cell ratio correlates with an increase in the number of CD4+ T cells. In some embodiments, the increase in the effector to suppressor T cell ratio correlates with an increase in the number of CD8+/CD4+ T cells. In some embodiments, the increase in the effector to suppressor T cell ratio correlates with a decrease in the number of Treg cells.
  • the effector to suppressor T cell ratio e.g. , the CD8 + T cell to Treg cell ratio, following administration of a polynucleotide encoding a tethered IL- 12 polypeptide (alone or in combination with an anti-PD-Ll antibody, an anti-PD- 1 antibody, and/or an anti-CTLA-4 antibody or a polynucleotide encoding the same) is at least about 1.5: 1, at least about 2: 1, at least about 2.5: 1, at least about 3: 1, at least about 3.5: 1, at least about 3.5: 1, at least about 4: 1, at least about 4.5: 1, at least about 5: 1, at least about 6: 1, at least about 7: 1, at least about 8: 1, at least about 9: 1, at least about 10: 1, at least about 15: 1, at least about 20: 1, at least about 25: 1, at least about 30: 1, at least about 35: 1, at least about 40: 1, at least about 45: 1, at least about 50: 1, at least about 60: 1,
  • the effector to suppressor T cell ratio e.g. , the CD8 + T cell to Treg cell ratio, following administration of a polynucleotide encoding a tethered IL- 12 polypeptide (alone or in combination with an anti-PD-Ll antibody, an anti-PD- 1 antibody, and/or an anti-CTLA-4 antibody or a polynucleotide encoding the same) is at least about 1.5, at least about 2, at least about 2.5, at least about 3, at least about 3.5, at least about 3.5, at least about 4, at least about 4.5, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at
  • the increase in the effector to suppressor T cell ratio in the tumor is directed to an anti-tumor immune response in the subject.
  • the increase in the effector to suppressor T cell ratio in the tumor reduces or decreases the size of a tumor or inhibits the growth of a tumor in the subject.
  • the effector to suppressor T cell ratio in the tumor can be measured using applications in the art such as measuring the ratio of CD8+, CD4+, or CD8+/CD4+ T cells to Treg cells, using any methods known in the art including IHC and/or flow cytometry.
  • the delivery of the polynucleotide encoding a tethered IL- 12 polypeptide to a tumor using a pharmaceutical composition for intratumoral administration disclosed herein can:
  • (c) decrease leakage of the polynucleotide or expressed product to off-target tissue (e.g., peritumoral tissue, or to distant locations, e.g., liver tissue); or,
  • off-target tissue e.g., peritumoral tissue, or to distant locations, e.g., liver tissue
  • a corresponding reference composition e.g., composition in which compounds of formula (I) are not present or have been substituted by another ionizable amino lipid, e.g., MC3
  • a decrease in leakage can be quantified as decrease in the ratio of polypeptide expression in the tumor to polypeptide expression in non-tumor tissues, such as peritumoral tissue or to another tissue or organ, e.g., liver tissue.
  • Delivery of a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide to a tumor involves administering a pharmaceutical composition disclosed herein, e.g., in nanoparticle form, including the polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide to a subject, where administration of the pharmaceutical composition involves contacting the tumor with the
  • a translatable mRNA upon contacting a cell in the tumor with the pharmaceutical composition, a translatable mRNA may be translated in the cell to produce a polypeptide of interest.
  • mRNAs that are substantially not translatable may also be delivered to tumors.
  • Substantially non-translatable mRNAs may be useful as vaccines and/or may sequester translational components of a cell to reduce expression of other species in the cell.
  • the pharmaceutical compositions disclosed herein can increase specific delivery.
  • specific delivery means delivery of more (e.g., at least 1.5 fold more, at least 2- fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, or at least 10-fold more) of a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide by pharmaceutical composition disclosed herein (e.g., in nanoparticle form) to a target tissue of interest (e.g., a tumor) compared to an off-target tissue (e.g., mammalian liver).
  • a target tissue of interest e.g., a tumor
  • off-target tissue e.g., mammalian liver
  • the level of delivery of a nanoparticle to a particular tissue may be measured, for example, by comparing
  • Specific delivery to a tumor or a particular class of cells in the tumor implies that a higher proportion of pharmaceutical composition including a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide is delivered to the target destination (e.g., target tissue) relative to other off-target destinations upon administration of a pharmaceutical composition to a subject.
  • the present disclosure also provides methods to deliver intratumorally a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide when a pharmaceutical composition comprising the polynucleotides disclosed herein (e.g., in nanoparticle form) are administered to a tumor.
  • the intratumoral administration can show one or more properties selected from:
  • a corresponding reference composition e.g., composition in which compounds of formula (I) are not present or have been substituted by another ionizable amino lipid, e.g., MC3
  • a decrease in leakage can be quantified as decrease in the ratio of polypeptide expression in the tumor to polypeptide expression in non-tumor tissues, such as peritumoral tissue or to another tissue or organ, e.g., liver tissue.
  • another improvement in delivery caused as a result of using the pharmaceutical compositions disclosed herein is a reduction in immune response with respect to the immune response observed when other lipid components are used to deliver the same a therapeutic agent or polynucleotide encoding a therapeutic agent.
  • the present disclosure provides a method of increasing retention of a therapeutic agent (e.g., a polypeptide administered as part of the pharmaceutical composition) in a tumor tissue in a subject, comprising administering intratumorally to the tumor tissue a pharmaceutical composition disclosed herein, wherein the retention of the therapeutic agent in the tumor tissue is increased compared to the retention of the therapeutic agent in the tumor tissue after administering a corresponding reference composition.
  • a therapeutic agent e.g., a polypeptide administered as part of the pharmaceutical composition
  • Also provided is a method of increasing retention of an expressed polypeptide in a tumor tissue in a subject comprising administering to the tumor tissue a pharmaceutical composition disclosed herein, wherein the pharmaceutical composition comprises a polynucleotide encoding the expressed polypeptide, and wherein the retention of the expressed polypeptide in the tumor tissue is increased compared to the retention of the polypeptide in the tumor tissue after administering a corresponding reference composition.
  • the present disclosure also provides a method of decreasing expression leakage of a polynucleotide administered intratumorally to a subject in need thereof, comprising administering the polynucleotide intratumorally to the tumor tissue as a pharmaceutical composition disclosed herein, wherein the expression level of the polypeptide in non-tumor tissue is decreased compared to the expression level of the polypeptide in non-tumor tissue after administering a corresponding reference composition.
  • a therapeutic agent e.g., a polypeptide administered as part of the pharmaceutical composition
  • Also provided is a method of decreasing expression leakage of an expressed polypeptide in a tumor in a subject comprising administering to the tumor tissue a pharmaceutical composition disclosed herein, wherein the pharmaceutical composition comprises a polynucleotide encoding the expressed polypeptide, and wherein the amount of expressed polypeptide in non-tumor tissue is decreased compared to the amount of expressed polypeptide in non-tumor tissue after administering a corresponding reference composition.
  • the non-tumoral tissue is peritumoral tissue. In other embodiments, the non-tumoral tissue is liver tissue.
  • the present disclosure also provides a method to reduce or prevent the immune response caused by the intratumoral administration of a pharmaceutical composition, e.g., a pharmaceutical composition comprising lipids known in the art, by replacing one or all the lipids in such composition with a compound of Formula (I).
  • a pharmaceutical composition e.g., a pharmaceutical composition comprising lipids known in the art
  • the immune response caused by the administration of a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide in a pharmaceutical composition comprising MC3 (or other lipids known in the art) can be prevented (avoided) or ameliorated by replacing MC3 with a compound of Formula (I), e.g., Compound 18.
  • the immune response observed after a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide is administered in a pharmaceutical composition disclosed herein is not elevated compared to the immune response observed when the therapeutic agent or a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide is administered in phosphate buffered saline (PBS) or another physiological buffer solution (e.g., Ringer's solution, Tyrode's solution, Hank's balanced salt solution, etc.).
  • PBS phosphate buffered saline
  • another physiological buffer solution e.g., Ringer's solution, Tyrode's solution, Hank's balanced salt solution, etc.
  • the immune response observed after a therapeutic agent or a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide is administered in a therapeutic agent or a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide is administered in a therapeutic agent or a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide is administered in a
  • composition disclosed herein is not elevated compared to the immune response observed when PBS or another physiological buffer solution is administered alone.
  • no immune response is observed when a pharmaceutical composition disclosed herein is administered intratumorally to a subject.
  • the present disclosure also provides a method of delivering a therapeutic agent or a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide to a subject in need thereof, comprising administering intratumorally to the subject a pharmaceutical composition disclosed herein, wherein the immune response caused by the administration of the pharmaceutical
  • composition is not elevated compared to the immune response caused by the intratumoral administration of
  • PBS alone or another physiological buffer solution (e.g., Ringer's solution, Tyrode's solution, Hank's balanced salt solution, etc.);
  • another physiological buffer solution e.g., Ringer's solution, Tyrode's solution, Hank's balanced salt solution, etc.
  • the therapeutic agent or polynucleotide e.g., mRNA, encoding a tethered IL-12 polypeptide in PBS or another physiological buffer solution; or,
  • a corresponding reference composition i.e., the same pharmaceutical composition in which the compound of Formula (I) is substituted by another ionizable amino lipid, e.g., MC3.
  • the administration treats a cancer.
  • the polynucleotide (e.g. , mRNA) of the present disclosure can be administered in any route available, including, but not limited to, intratumoral, enteral, gastroenteral, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraperitoneal (into the peritoneum), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection, (into the base of the penis),
  • the polynucleotide, e.g., mRNA, of the present disclosure is administered parenterally (e.g. , includes subcutaneous, intravenous, intraperitoneal, intratumoral, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques), intraventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenterally e.g. , includes subcutaneous, intravenous, intraperitoneal, intratumoral, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques
  • intraventricularly orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the polynucleotide, composition, or polypeptide is administered subcutaneously, intravenously, intraperitoneally, intratumorally, intramuscularly, intra-articularly, intra- synovially, intrasternally, intrathecally, intrahepatically, intradermally, intralesionally, intracranially, intraventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the polynucleotide (e.g. , mRNA) of the present disclosure is administered intratumorally.
  • the polynucleotide e.g., mRNA
  • a device comprising a pump, patch, drug reservoir, short needle device, single needle device, multiple needle device, micro-needle device, jet injection device, ballistic powder/particle delivery device, catheter, lumen, cryoprobe, cannula, microcanular, or devices utilizing heat, RF energy, electric current, or any combination thereof.
  • aspects of the present disclosure relate to transplantation of cells containing polynucleotides to a mammalian subject.
  • Administration of cells to mammalian subjects is known to those of ordinary skill in the art, and includes, but is not limited to, local implantation (e.g., topical or subcutaneous administration), organ delivery or systemic injection (e.g., intravenous injection or inhalation), and the formulation of cells in pharmaceutically acceptable carriers.
  • the polynucleotide e.g., mRNA
  • the administration of the polynucleotide, e.g., mRNA, pharmaceutical composition or formulation of the disclosure results in expression of IL-12 in cells of the subject.
  • administering the polynucleotide, e.g., mRNA, pharmaceutical composition or formulation of the disclosure results in an increase of IL-12 activity in the subject.
  • the polynucleotides of the present disclosure are used in methods of
  • compositions or formulation comprising an mRNA encoding a tethered IL- 12 polypeptide to a subject, wherein the method results in an increase of IL- 12 activity in at least some cells of a subject.
  • the administration of a composition or formulation comprising an mRNA encoding a tethered IL-12 polypeptide to a subject results in an increase of IL- 12 activity in cells subject to a level at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or to 100% or more of the activity level expected in a normal subject.
  • inventions of the disclosure also provide a method of treating a cancer in a subject in need thereof comprising administering (e.g. , intratumorally, intraperitoneally, or intravenously) a polynucleotide comprising an mRNA encoding a tethered IL- 12 polypeptide with one or more anticancer agents to the subject.
  • administering e.g. , intratumorally, intraperitoneally, or intravenously
  • a polynucleotide comprising an mRNA encoding a tethered IL- 12 polypeptide with one or more anticancer agents to the subject.
  • the polynucleotides e.g. , mRNA
  • a tethered IL- 12 polypeptide of the present disclosure can be used to reduce or decrease the size of a tumor or inhibit growth of a tumor in a subject in need thereof.
  • the tumor is associated with a disease, disorder, and/or condition.
  • the disease, disorder, and/or condition is a cancer.
  • the administration of the polynucleotide (e.g. , mRNA) encoding a tethered IL- 12 polypeptide treats a cancer.
  • a “cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream.
  • a “cancer” or “cancer tissue” can include a tumor at various stages.
  • the cancer or tumor is stage 0, such that, e.g. , the cancer or tumor is very early in development and has not metastasized.
  • the cancer or tumor is stage I, such that, e.g. , the cancer or tumor is relatively small in size, has not spread into nearby tissue, and has not metastasized.
  • the cancer or tumor is stage II or stage III, such that, e.g. , the cancer or tumor is larger than in stage 0 or stage I, and it has grown into neighboring tissues but it has not metastasized, except potentially to the lymph nodes.
  • the cancer or tumor is stage IV, such that, e.g. , the cancer or tumor has metastasized. Stage IV can also be referred to as advanced or metastatic cancer.
  • the cancer can include, but is not limited to, adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor
  • the tumor is a solid tumor.
  • a “solid tumor” includes, but is not limited to, sarcoma, melanoma, carcinoma, or other solid tumor cancer.
  • Sparcoma refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sar
  • melanoma refers to a tumor arising from the melanocytic system of the skin and other organs.
  • Melanomas include, for example, acra-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, metastatic melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas include, e.g. , acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere
  • Additional cancers that can be treated include, e.g. , Leukemia, Hodgkin's Disease, Non- Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, papillary thyroid cancer, neuroblastoma, neuroendocrine cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, prostate cancer, Miillerian cancer, ovarian cancer, peritoneal cancer, fallopian tube cancer, or uterine papillary serous carcinoma.
  • Leukemia e.g
  • the disclosure further includes a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide or uses thereof as a combination therapy, i.e. , with any other anti-cancer agent in combination.
  • a polynucleotide e.g., mRNA
  • encoding a tethered IL- 12 polypeptide or uses thereof as a combination therapy i.e. , with any other anti-cancer agent in combination.
  • the disclosure is directed to a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide in combination with one or more anti-cancer agents or uses of the polynucleotide in combination with one or more anti-cancer agents to the subject.
  • a polynucleotide e.g., mRNA
  • the combination therapy can be a combination of the polynucleotide, e.g., mRNA, encoding IL- 12 and one or more standard therapy.
  • the methods of the disclosure include two additional anti-cancer agents, three additional agents, four additional agents, etc.
  • the additional anti-cancer agents can be a protein, e.g., an antibody, or a polynucleotide, e.g., mRNA.
  • the one or more anti-cancer agents are an mRNA.
  • the one or more anti-cancer agents are a polynucleotide encoding a tumor antigen.
  • the one or more anti-cancer agents are an mRNA encoding a tumor antigen. In other embodiments, the one or more anti-cancer agents are not a tumor antigen or an mRNA encoding a tumor antigen. In other embodiments, the one or more anti-cancer agents are a protein, e.g., an antibody.
  • the one or more anti-cancer agents are an approved agent by the United States Food and Drug Administration. In other embodiments, the one or more anti-cancer agents are a pre-approved agent by the United States Food and Drug Administration.
  • alternative embodiments of the present disclosure include a combination therapy of a polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide and any other agents, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody.
  • a polynucleotide e.g., mRNA
  • any other agents e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, and/or an anti-CTLA-4 antibody.
  • the present disclosure encompasses combination therapy of (i) a polynucleotide (e.g., mRNA) encoding a tethered IL- 12 polypeptide and a protein comprising an anti-PD- 1 antibody or an anti-PD-Ll antibody; or (iii) a polynucleotide (e.g., mRNA) encoding a tethered IL- 12 polypeptide and a second protein comprising an anti-CTLA-4 antibody.
  • a polynucleotide e.g., mRNA
  • a polynucleotide e.g., mRNA
  • the additional agents can be formulated together with the
  • polynucleotide encoding a tethered IL- 12 polypeptide, e.g., mRNA, or separately.
  • the additional agents can be administered concurrently with the polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide or sequentially.
  • the polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide is administered prior to the second agent.
  • the polynucleotide, e.g., mRNA, encoding a tethered IL- 12 polypeptide is administered after the second agent.
  • the additional agents e.g., any antibody disclosed herein
  • the second agents e.g., any antibody disclosed herein
  • the subject for the present methods or compositions has been treated with one or more standard of care therapies. In other aspects, the subject for the present methods or compositions has not been responsive to one or more standard of care therapies or anti-cancer therapies.
  • the subject has been previously treated with an IL- 12 protein or an IL- 12 DNA gene therapy.
  • the subject is treated with an anti-PD-1 antagonist prior to the polynucleotide of the present disclosure.
  • the subject has been treated with a monoclonal antibody that binds to PD-1 prior to the polynucleotide of the present disclosure.
  • the subject has been treated with an anti-PD-1 monoclonal antibody therapy prior to the polynucleotide of the present methods or compositions.
  • T cell regulation i.e., activation or inhibition
  • activating receptors such as OX40
  • inhibitory receptors i.e., immune checkpoints
  • PD-1 programmed death receptor 1
  • CLA-4 cytotoxic T lymphocyte-associated protein 4
  • Immune checkpoint inhibitors such as pembrolizumab or nivolumab, which target the interaction between programmed death receptor 1/programmed death ligand 1 (PD-1/PD-L1) and PD-L2, have been recently approved for the treatment of various malignancies and are currently being investigated in clinical trials for cancers including melanoma, head and neck squamous cell carcinoma (HNSCC). Data available from these trials indicate substantial activity accompanied by a favorable safety and toxicity profile in these patient populations.
  • HNSCC head and neck squamous cell carcinoma
  • checkpoint inhibitors have been tested in clinical trials for the treatment of melanoma.
  • phase III clinical trials have revealed that therapies such as ipilimumab and pembrolizumab, which target the CTLA-4 and PD-1 immune checkpoints, respectively, have raised the three-year survival of patients with melanoma to ⁇ 70%, and overall survival (>5years) to ⁇ 30%.
  • checkpoint inhibitors have been tested in clinical trials for the treatment of head and neck cancer.
  • PD-L1 programmed death ligand 1
  • the subject has been previously treated with a PD-1 antagonist prior to the polynucleotide of the present disclosure.
  • the subject has been treated with a monoclonal antibody that binds to PD-1 prior to the polynucleotide of the present disclosure.
  • the subject has been treated with an anti-PD-1 monoclonal antibody therapy prior to the polynucleotide of the present methods or compositions.
  • the anti-PD- 1 monoclonal antibody therapy comprises nivolumab, pembrolizumab, pidilizumab, or any combination thereof.
  • the subject has been treated with a monoclonal antibody that binds to PD-Ll prior to the polynucleotide of the present disclosure.
  • the subject has been treated with an anti-PD-Ll monoclonal antibody therapy prior to the polynucleotide of the present methods or compositions.
  • the anti-PD-Ll monoclonal antibody therapy comprises durvalumab, avelumab, MEDI473, BMS-936559, aezolizumab, or any combination thereof.
  • the subject has been treated with a CTLA-4 antagonist prior to treatment with the compositions of present disclosure.
  • the subject has been previously treated with a monoclonal antibody that binds to CTLA-4 prior to the compositions of the present disclosure.
  • the subject has been treated with an anti-CTLA-4 monoclonal antibody prior to the polynucleotide of the present disclosure.
  • the anti-CTLA-4 antibody therapy comprises ipilimumab or tremelimumab.
  • the disclosure is directed to a method of treating cancer and/or a method of immunotherapy in a subject in need thereof comprising administering to the subject (e.g. , intratumorally, intraperitoneally, or intravenously) the polynucleotide (e.g., RNA, e.g., mRNA) encoding a tethered IL- 12 polypeptide in combination with a PD-Ll antagonist, e.g., an antibody or antigen-binding portion thereof that specifically binds to PD-Ll, e.g., an anti-PD-Ll monoclonal antibody, e.g., an anti-PD-Ll monoclonal antibody comprises Durvalumab, Avelumab, MEDI473, BMS-936559, Atezolizumab, or any combination thereof.
  • the polynucleotide e.g., RNA, e.g., mRNA
  • a PD-Ll antagonist e.g
  • the anti-PD-Ll antibody useful for the disclosure is MSB0010718C (also called Avelumab; See US 2014/0341917) or BMS-936559 (formerly 12A4 or MDX-1105) (see, e.g., U.S. Patent No. 7,943,743; WO 2013/173223).
  • the anti-PD-Ll antibody is MPDL3280A (also known as RG7446) (see, e.g., Herbst et al. (2013) Clin Oncol 31(suppl):3000. Abstract; U.S. Patent No. 8,217,149), MEDI4736 (also called Durvalumab; Khleif (2013) In: Proceedings from the European Cancer Congress 2013; September 27-October 1, 2013; Amsterdam, The Netherlands.
  • the disclosure is directed to a method of treating cancer and/or a method of immunotherapy in a subject in need thereof comprising administering to the subject ⁇ e.g., intratumorally, intraperitoneally, or intravenously) the polynucleotide ⁇ e.g., RNA, e.g., mRNA) encoding a tethered IL-12 polypeptide, in combination with a PD-1 antagonist, e.g., an antibody or antigen -binding portion thereof that specifically binds to PD-1, e.g., an anti-PD-1 monoclonal antibody.
  • a PD-1 antagonist e.g., an antibody or antigen -binding portion thereof that specifically binds to PD-1, e.g., an anti-PD-1 monoclonal antibody.
  • the anti-PD-1 antibody (or an antigen-binding portion thereof) useful for the disclosure is pembrolizumab.
  • Pembrolizumab also known as "KEYTRUDA ® ", lambrolizumab, and MK-3475
  • KEYTRUDA ® a humanized monoclonal IgG4 antibody directed against human cell surface receptor PD-1 (programmed death- 1 or programmed cell death- 1).
  • Pembrolizumab has been approved by the FDA for the treatment of relapsed or refractory melanoma and advanced NSCLC.
  • the anti-PD-1 antibody useful for the disclosure is nivolumab.
  • Nivolumab also known as "OPDIVO ® "; formerly designated 5C4, BMS-936558, MDX-1106, or ONO-4538
  • OPDIVO ® is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the down- regulation of antitumor T-cell functions
  • Nivolumab has shown activity in a variety of advanced solid tumors including renal cell carcinoma (renal adenocarcinoma, or hypernephroma), melanoma, and non- small cell lung cancer (NSCLC) (Topalian et al., 2012a; Topalian et al., 2014; Drake et al., 2013; WO 2013/173223.
  • the anti-PD- 1 antibody is MED 10680 (formerly AMP-514), which is a monoclonal antibody against the PD- 1 receptor.
  • the anti-PD-1 antibody is BGB-A317, which is a humanized monoclonal antibody.
  • BGB-A317 is described in U.S. Publ. No. 2015/0079109.
  • a PD-1 antagonist is AMP-224, which is a B7-DC Fc fusion protein.
  • the disclosure includes a method of treating cancer and/or a method of immunotherapy in a subject in need thereof comprising administering to the subject (e.g. , intratumorally, intraperitoneally, or intravenously) the polynucleotide (e.g., RNA, e.g., mRNA) encoding a tethered IL- 12 polypeptide together with an antibody or an antigen binding portion thereof that specifically binds to PD-1, e.g., an anti-PD- 1 monoclonal antibody, e.g., an anti-PD- 1 monoclonal antibody comprises Nivolumab, Pembrolizumab, Pidilizumab, or any combination thereof.
  • the polynucleotide e.g., RNA, e.g., mRNA
  • an antigen binding portion thereof that specifically binds to PD-1
  • an anti-PD- 1 monoclonal antibody e.g., an anti-PD- 1 monoclon
  • the disclosure is directed to a method of treating cancer and/or a method of immunotherapy in a subject in need thereof comprising administering to the subject (e.g. , intratumorally, intraperitoneally, or intravenously) the polynucleotide (e.g., RNA, e.g., mRNA) encoding a tethered IL- 12 polypeptide in combination with a CTLA-4 antagonist, e.g., an antibody or antigen-binding portion thereof that specifically binds to CTLA-4, e.g., an anti-CTLA-4 monoclonal antibody, e.g., an anti-CTLA-4 monoclonal antibody comprises Ipilimumab or
  • Tremelimumab or any combination thereof.
  • An exemplary clinical anti-CTLA-4 antibody is the human mAb 10D 1 (now known as ipilimumab and marketed as YERVOY®) as disclosed in U.S. Patent No. 6,984,720.
  • Another anti- CTLA-4 antibody useful for the present methods is tremelimumab (also known as CP-675,206).
  • Tremelimumab is human IgG2 monoclonal anti-CTLA-4 antibody. Tremelimumab is described in WO/2012/122444, U.S. Publ. No. 2012/263677, or WO Publ. No. 2007/113648 A2.
  • compositions disclosed herein comprise (i) a polynucleotide, e.g., mRNA, encoding a tethered IL-12 polypeptide and (ii) a polynucleotide, e.g., mRNA, encoding an antibody or an antigen binding portion thereof which specifically binds to CTLA-4 in a single formulation.
  • IL-12 (also shown as IL12) is a pleiotropic cytokine, the actions of which create an interconnection between innate and adaptive immunity.
  • IL-12 functions primarily as a 70 kDa heterodimeric protein consisting of two disulfide-linked p35 and p40 subunits.
  • the precursor form of the IL-12 p40 subunit (NM_002187; P29460; also referred to as IL-12B, natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2) is 328 amino acids in length, while its mature form is 306 amino acids long.
  • the precursor form of the IL-12 p35 subunit (NM_000882; P29459; also referred to as IL-12A, natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1) is 219 amino acids in length and the mature form is 197 amino acids long.
  • the genes for the IL-12 p35 and p40 subunits reside on different chromosomes and are regulated independently of each other. Gately, MK et al., Annu Rev Immunol. 16: 495-521 (1998). Many different immune cells ⁇ e.g., dendritic cells, macrophages, monocytes, neutrophils, and B cells) produce IL-12 upon antigenic stimuli.
  • the active IL-12 heterodimer is formed following protein synthesis. Id.
  • IL-12 is composed of a bundle of four alpha helices. It is a heterodimeric cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40).
  • the active heterodimer (referred to as ' ⁇ 70'), and a homodimer of p40 are formed following protein synthesis.
  • the tethered IL-12 polypeptide of the present disclosure comprises IL- 12A. In some embodiments, the tethered IL-12 polypeptide of the present disclosure comprises IL- 12B. In some embodiments, the tethered IL-12 polypeptide of the present disclosure comprises both IL-12A and IL-12B.
  • IL-12B is located N-terminal to IL-12A in the tethered IL-12 polypeptide of the present disclosure.
  • IL-12A is located N-terminal to IL-12B in the tethered IL-12 polypeptide of the present disclosure.
  • the phrase "located N-terminal to” indicates location in a polypeptide with respect to other sequences in the polypeptide in relation to the N-terminus of the polypeptide.
  • IL-12B that is "N-terminal to" IL-12A means that IL-12B is located closer to the N-terminus of the tethered IL-12 polypeptide than IL-12A.
  • the tethered IL- 12 polypeptide of the present disclosure comprises a single polypeptide chain comprising IL- 12B and IL-12A, which are fused directly to one another or are linked to one another by a linker (referred to herein as an "subunit linker").
  • linkers are disclosed elsewhere herein.
  • the tethered IL- 12 polypeptide of the disclosure comprises IL-12A and/or IL-12B that is a variant, that is a functional fragment, or that contains a substitution, an insertion and/or an addition, a deletion, and/or a covalent modification with respect to a wild-type IL- 12A or IL- 12B sequence.
  • sequence tags such as epitope tags, e.g. , a V5 tag
  • amino acids can be added to the sequences encoded by the polynucleotides of the disclosure (e.g., at the N-terminal or C-terminal ends), e.g., for localization.
  • amino acid residues located at the carboxy, amino terminal, or internal regions of a polypeptide of the disclosure can optionally be deleted providing for fragments.
  • the tethered IL- 12 polypeptide encoded by a polynucleotide of the disclosure comprises a substitutional variant of an IL- 12A and/or IL- 12B sequence, which can comprise one, two, three or more than three substitutions.
  • the substitutional variant can comprise one or more conservative amino acids substitutions.
  • the variant is an insertional variant.
  • the variant is a deletional variant.
  • IL-12 protein fragments, functional protein domains, variants, and homologous proteins are also considered to be within the scope of the IL- 12 polypeptides of the disclosure.
  • Nonlimiting examples of polypeptides encoded by the polynucleotides of the disclosure are set forth in SEQ ID NOs: 1, 3, 45, 48 and 49. .
  • SEQ ID NOs: 1, 3, and 45 provide the amino acid sequence of human wild type IL- 12.
  • the tethered IL- 12 polypeptides of the disclosure comprise a membrane domain that tethers (i.e. , anchors) the IL- 12 polypeptide to a cell membrane (e.g. , a transmembrane domain).
  • the tethered IL- 12 polypeptides comprise a transmembrane domain.
  • the tethered IL- 12 polypeptides comprise a transmembrane domain, and optionally an intracellular domain.
  • the tethered IL-12 polypeptides comprise a membrane domain that tethers (i.e. , anchors) the IL- 12 polypeptide to a cell membrane (e.g. , a transmembrane domain).
  • the tethered IL- 12 polypeptides comprise a transmembrane domain.
  • the tethered IL- 12 polypeptides comprise a transmembrane domain, and optionally an intracellular domain.
  • transmembrane domain and an intracellular domain In some embodiments, the membrane domain is from an integral membrane protein.
  • Integral membrane proteins can include, for example, integral polytopic proteins that contain a single-pass or multi-pass transmembrane domain that tethers the protein to a cell surface, including domains with hydrophobic a-helical or ⁇ -barrel (i.e. , ⁇ -sheet) structures.
  • the amino-terminus (i.e. , N-terminus) of Type I integral membrane proteins is located in the extracellular space, while the carboxy-terminus (i.e. , C-terminus) of Type II integral membrane proteins is located in the intracellular space.
  • a tethered IL- 12 polypeptide of the disclosure comprises a
  • a tethered IL-12 polypeptide of the disclosure comprises a transmembrane domain from a Type I integral membrane protein.
  • a tethered IL- 12 polypeptide comprises a transmembrane domain from a Type II integral membrane protein.
  • the transmembrane domain comprises an intracellular domain (i.e., a domain that is localized to the intracellular space of a cell, e.g. , a domain that is localized to the cytoplasm of a cell). In some embodiments, an intracellular domain has been removed from the transmembrane domain. In some embodiments, the transmembrane domain comprises a membrane domain without an intracellular domain.
  • Integral membrane proteins can also include, for example, integral monotopic proteins that contain a membrane domain that does not span the entire cell membrane but that tethers the protein to a cell surface.
  • a tethered IL- 12 polypeptide of the disclosure comprises a membrane domain from an integral monotopic protein.
  • the membrane domain is from a Cluster of Differentiation (CD) protein, CD8, CD80, CD4, a receptor, Platelet-Derived Growth Factor Receptor (PDGF-R), Interleukin-6 Receptor (IL-6R), transferrin receptor, Tumor Necrosis Factor (TNF) receptor, erythropoietin (EPO) receptor, a T Cell Receptor (TCR), TCR ⁇ -chain, a Fc receptor, FcyRII, FcsRI, an interferon receptor, type I interferon receptor, a growth factor, Stem Cell Factor (SCF), TNF-a, B7-1, Asialoglycoprotein, c-erbB-2, ICAM-1, an immunoglobulin, an IgG, an IgM, a viral glycoprotein, rabies virus glycoprotein, respiratory syncytial virus glycoprotein G (RSVG), vesicular stomatis virus glycoprotein (VSVG), a viral hemagglu
  • transmembrane domains are set forth in SEQ ID NOs: 101- 103.
  • a membrane domain comprises a transmembrane domain of T-cell surface glycoprotein CD8 alpha chain (also known as CD8A or T-lymphocyte differentiation antigen T8/Leu-2), e.g., a transmembrane of UniProtKB - P01732.
  • CD8A T-cell surface glycoprotein CD8 alpha chain
  • T8/Leu-2 T-lymphocyte differentiation antigen T8/Leu-2
  • polynucleotide e.g., mRNA, encoding a tethered IL- 12
  • polynucleotide comprises a nucleotide sequence encoding a CD8 transmembrane polypeptide.
  • the polynucleotide e.g., mRNA, encoding a tethered IL-12
  • a membrane domain comprises a transmembrane domain of platelet- derived growth factor receptor beta (EC:2.7.10.1) (also known as PDGF-R-beta, PDGFR-beta, beta platelet-derived growth factor receptor, beta-type platelet-derived growth factor receptor, CD 140 antigen-like family member B, platelet-derived growth factor receptor 1, PDGFR- 1, or CD140b), e.g., a transmembrane domain of UniProtKB - P09619.
  • platelet- derived growth factor receptor beta EC:2.7.10.1
  • PDGF-R-beta also known as PDGF-R-beta, PDGFR-beta, beta platelet-derived growth factor receptor, beta-type platelet-derived growth factor receptor, CD 140 antigen-like family member B, platelet-derived growth factor receptor 1, PDGFR- 1, or CD140b
  • the polynucleotide e.g., mRNA, encoding a tethered IL- 12, comprises a nucleotide sequence encoding a PDGFR-beta transmembrane polypeptide.
  • the polynucleotide, e.g., mRNA, encoding a tethered IL- 12 comprises a nucleotide sequence encoding a PDGFR-beta transmembrane
  • polypeptide as set forth in SEQ ID NO: 102.
  • a membrane domain comprises a transmembrane domain of T- lymphocyte activation antigen CD80 (also known as activation B7- 1 antigen, BB l, CTLA-4 counter- receptor B7.1, or B7), e.g., a transmembrane domain of UniProtKB - P33681.
  • CD80 also known as activation B7- 1 antigen, BB l, CTLA-4 counter- receptor B7.1, or B7
  • the polynucleotide e.g., mRNA, encoding a tethered IL- 12, comprises a nucleotide sequence encoding a CD80 transmembrane polypeptide.
  • the polynucleotide, e.g., mRNA, encoding a tethered IL- 12 comprises a nucleotide sequence encoding a CD80 transmembrane polypeptide as set forth in SEQ ID NO: 103.
  • the membrane domain in the tethered IL- 12 polypeptide comprises an amino acid sequence at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical to SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, or any combination thereof.
  • the membrane domain in the tethered IL- 12 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 101, SEQ ID NO: 102, or SEQ ID NO: 103 without one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, or nine amino acids at the N terminus of SEQ ID NO: 101, SEQ ID NO: 102, or SEQ ID NO: 103 and/or without one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, or nine amino acids at the C terminus of SEQ ID NO: 101, SEQ ID NO: 102, or SEQ ID NO: 103.
  • the membrane domain in the tethered IL- 12 polypeptide comprises SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, or any combination thereof with one amino acid substitution, two amino acid substitutions, three amino acid substitutions, four amino acid substitutions, five amino acid substitutions, six amino acid substitutions, seven amino acid substitutions, eight amino acid substitutions, or nine amino acid substitutions, wherein the membrane domain is capable of tethering the IL- 12 polypeptide on a cell membrane.
  • the membrane domain comprises a transmembrane domain and an intracellular domain.
  • an intracellular domain is any oligopeptide or polypeptide known to act as a transmission signal in a cell.
  • the membrane domain comprises an intracellular domain to stabilize the tethered IL- 12 polypeptide.
  • Intracellular domains useful in the methods and compositions of the present disclosure include at least those derived from any of the polypeptides in which transmembrane domains are derived, as described supra.
  • suitable intracellular domains include, but are not limited to, an intracellular domain derived from CD80, PDGFR, or any combination thereof.
  • a membrane domain comprises an intracellular domain of platelet- derived growth factor receptor beta (EC:2.7.10.1) (also known as PDGF-R-beta, PDGFR-beta, beta platelet-derived growth factor receptor, beta-type platelet-derived growth factor receptor, CD 140 antigen-like family member B, platelet-derived growth factor receptor 1, PDGFR- 1, or CD140b), e.g., an intracellular domain of UniProtKB - P09619.
  • the polynucleotide e.g., mRNA, encoding a tethered IL-12, comprises a nucleotide sequence encoding a PDGFR-beta intracellular polypeptide.
  • the polynucleotide e.g., mRNA, encoding a tethered IL- 12, comprises a nucleotide sequence encoding a PDGFR-beta intracellular polypeptide as set forth in SEQ ID NO: 226.
  • a membrane domain comprises a truncated intracellular domain of PDGFR-beta.
  • a truncated intracellular domain of PDGFR-beta stabilizes the tethered IL- 12 polypeptide compared to the wild-type PDGFR-beta intracellular domain.
  • the polynucleotide e.g., mRNA, encoding a tethered IL- 12, comprises a nucleotide sequence encoding a truncated PDGFR-beta intracellular polypeptide.
  • the polynucleotide, e.g., mRNA, encoding a tethered IL- 12 comprises a nucleotide sequence encoding a truncated PDGFR-beta intracellular polypeptide as set forth in SEQ ID NO: 227.
  • the polynucleotide e.g., mRNA, encoding a tethered IL- 12, comprises a nucleotide sequence encoding a truncated PDGFR-beta intracellular polypeptide as set forth in SEQ ID NO: 228.
  • a membrane domain comprises an intracellular domain of T- lymphocyte activation antigen CD80 (also known as activation B7- 1 antigen, BB l, CTLA-4 counter- receptor B7.1, or B7), e.g., an intracellular domain of UniProtKB - P33681.
  • the polynucleotide e.g., mRNA, encoding a tethered IL-12, comprises a nucleotide sequence encoding a CD80 intracellular polypeptide.
  • the polynucleotide e.g., mRNA, encoding a tethered IL- 12, comprises a nucleotide sequence encoding a CD 80 intracellular polypeptide as set forth in SEQ ID NO: 225.
  • the tethered IL- 12 polypeptides described herein comprise a membrane domain comprising a transmembrane domain and an intracellular domain derived from the same polypeptide (i.e., homologous). In some embodiments, the tethered IL- 12 polypeptides described herein comprise a membrane domain comprising a CD80 transmembrane domain and CD80 intracellular domain. In some embodiments, the tethered IL-12 polypeptide described herein comprise a membrane domain comprising a PDGFR-beta transmembrane domain and PDGFR-beta intracellular domain.
  • the tethered IL-12 polypeptides described herein comprise a membrane domain comprising a transmembrane domain and an intracellular domain derived from different polypeptides (i.e., heterologous) (e.g., a CD80 transmembrane domain and a PDGFR-beta intracellular domain; a CD8 transmembrane domain and a CD80 intracellular domain; a CD8 transmembrane domain and a PDGFR-beta transmembrane domain; or a PDGFR-beta transmembrane domain and a CD80 intracellular domain).
  • heterologous e.g., a CD80 transmembrane domain and a PDGFR-beta intracellular domain; a CD8 transmembrane domain and a CD80 intracellular domain; a CD8 transmembrane domain and a PDGFR-beta transmembrane domain and a CD80 intracellular domain.
  • the membrane domain (e.g., transmembrane domain, and optional intracellular domain) in the tethered IL- 12 polypeptide is located C-terminal to any IL-12 amino acid sequence (i.e. , any amino acid sequence of IL-12A, IL- 12B, or both IL- 12A and IL- 12B when both are present in the tethered IL- 12 polypeptide).
  • IL-12 amino acid sequence i.e. , any amino acid sequence of IL-12A, IL- 12B, or both IL- 12A and IL- 12B when both are present in the tethered IL- 12 polypeptide.
  • located C-terminal to indicates location in a polypeptide with respect to other sequences in the polypeptide in relation to the C-terminus of the polypeptide.
  • a membrane domain e.g., transmembrane domain, and optional intracellular domain
  • a membrane domain that is "C-terminal to" any IL- 12 amino acid sequences means that the membrane domain is located closer to the C-terminus of the tethered IL-12 polypeptide than any IL- 12 amino acid sequences.
  • the membrane domain (e.g., transmembrane domain, and optional intracellular domain) in the tethered IL- 12 polypeptide is from a Type I integral membrane protein and is located C-terminal to any IL- 12 amino acid sequence.
  • the membrane domain (e.g., transmembrane domain, and optional intracellular domain) in the tethered IL- 12 polypeptide is located N-terminal to the IL-12
  • a membrane domain that is "N-terminal to" any IL- 12 amino acid sequences means that the membrane domain is located closer to the N-terminus of the tethered IL- 12 polypeptide than any IL- 12 amino acid sequences.
  • the membrane domain (e.g., transmembrane domain, and optional intracellular domain) in the tethered IL- 12 polypeptide is from a Type II integral membrane protein and is located N-terminal to any IL- 12 amino acid sequence.
  • the membrane domain (e.g., transmembrane domain, and optional intracellular domain) in the tethered IL- 12 polypeptide is linked to the IL- 12 polypeptide by a linker, which is referred to herein as a "membrane domain linker" or a "transmembrane domain linker” when the membrane domain is a transmembrane domain, and optionally an intracellular domain.
  • linkers are disclosed elsewhere herein.
  • the membrane domain in the tethered IL- 12 polypeptide is fused directly to the IL- 12 polypeptide.
  • the disclosure provides polynucleotides (e.g., a RNA, e.g., an mRNA) that comprise a nucleotide sequence encoding a tethered IL-12 polypeptide.
  • a RNA e.g., an mRNA
  • the disclosure provides polynucleotides (e.g., a RNA, e.g., an mRNA) comprising a nucleic acid sequence encoding an IL-12 polypeptide and a nucleic acid sequence encoding a membrane domain (e.g., transmembrane domain, and optional intracellular domain), wherein the nucleic acid sequence encoding the membrane domain is fused directly to the nucleic acid sequence encoding the IL- 12 polypeptide or is linked to the nucleic acid sequence encoding the IL- 12 polypeptide by a nucleic acid sequence encoding a linker (membrane domain linker).
  • a RNA e.g., an mRNA
  • a membrane domain e.g., transmembrane domain, and optional intracellular domain
  • the disclosure provides polynucleotides (e.g., a RNA, e.g., an mRNA) comprising a nucleic acid sequence encoding an IL-12 polypeptide, and a nucleic acid sequence encoding a transmembrane domain, wherein the nucleic acid sequence encoding the transmembrane domain is fused directly to the nucleic acid sequence encoding the IL-12 polypeptide or is linked to the nucleic acid sequence encoding the IL-12 polypeptide by a nucleic acid sequence encoding a linker (membrane domain linker).
  • a linker membrane domain linker
  • the disclosure provides polynucleotides (e.g., a RNA, e.g., an mRNA) comprising a nucleic acid sequence encoding an IL-12 polypeptide, a nucleic acid sequence encoding a transmembrane domain, and a nucleic acid encoding an intracellular domain, wherein the nucleic acid sequence encoding the transmembrane domain is fused directly to the nucleic acid sequence encoding the IL-12 polypeptide or is linked to the nucleic acid sequence encoding the IL- 12 polypeptide by a nucleic acid sequence encoding a linker (membrane domain linker), and wherein the nucleic acid encoding the intracellular domain is fused directly to the nucleic acid sequence encoding the transmembrane domain, or is linked to the nucleic acid sequence encoding the transmembrane domain by a nucleic acid sequence encoding a linker.
  • a linker e.g.
  • the IL-12 polypeptide comprises an IL- 12 p40 subunit (IL- 12B), an IL- 12A p35 subunit (IL- 12A), or both.
  • the nucleic acid sequence encoding the membrane domain is located at the 3' terminus of the nucleic acid sequence encoding the IL- 12 polypeptide or at the 3' terminus of the nucleic acid sequence encoding the membrane domain linker. In some embodiments, the nucleic acid sequence encoding the membrane domain (e.g., transmembrane domain, and optional intracellular domain) is located at the 5' terminus of the nucleic acid sequence encoding the IL- 12 polypeptide or at the 5' terminus of the nucleic acid sequence encoding the membrane domain linker.
  • the polynucleotide comprises an open reading frame (ORF) comprising a nucleic acid sequence encoding IL- 12B, a nucleic acid sequence encoding IL- 12A, and a nucleic acid sequence encoding a membrane domain (e.g., transmembrane domain, and optional intracellular domain), wherein the ORF optionally comprises a nucleic acid sequence encoding a linker (subunit linker) that links the IL- 12B and the IL- 12A.
  • ORF open reading frame
  • the nucleic acid sequence encoding the IL-12B is located at the 5' terminus of the nucleic acid sequence encoding the IL-12A or at the 5' terminus of the nucleic acid sequence encoding the subunit linker.
  • the nucleic acid sequence encoding the IL-12B is located at the 3' terminus of the nucleic acid sequence encoding the IL-12A or at the 3' terminus of the nucleic acid sequence encoding the subunit linker.
  • the nucleic acid sequence encoding the membrane domain is located at the 3' terminus of the nucleic acid sequence encoding the IL- 12A or at the 3' terminus of the membrane domain linker.
  • the nucleic acid sequence encoding the membrane domain is located at the 5' terminus of the nucleic acid sequence encoding the IL- 12B or at the 5' terminus of the membrane domain linker.
  • the IL-12 polypeptide comprises an IL- 12B polypeptide (i.e. , IL- 12B) selected from:
  • a functional fragment of the wild-type IL-12B polypeptide e.g., a truncated (e.g., deletion of carboxy, amino terminal, or internal regions) sequence shorter than an IL-12B wild-type; but still retaining IL-12B enzymatic activity;
  • a variant thereof e.g., full length or truncated IL-12B proteins in which one or more amino acids have been replaced, e.g., variants that retain all or most of the IL- 12B activity of the polypeptide with respect to the wild type IL- 12B polypeptide (such as, e.g., V33I, V298F, or any other natural or artificial variants known in the art); and
  • a fusion protein comprising (i) a full length IL- 12B wild-type, a functional fragment or a variant thereof, and (ii) a heterologous protein.
  • the IL-12 polypeptide comprises an IL- 12A polypeptide (i.e. , IL- 12A) selected from:
  • the full-length IL-12A polypeptide e.g., having the same or essentially the same length as wild-type IL-12 A
  • a functional fragment of the wild-type IL-12A polypeptide e.g., a truncated (e.g., deletion of carboxy, amino terminal, or internal regions) sequence shorter than an IL-12A wild-type; but still retaining IL-12A enzymatic activity;
  • a variant thereof e.g., full length or truncated IL-12A proteins in which one or more amino acids have been replaced, e.g., variants that retain all or most of the IL- 12A activity of the polypeptide with respect to the wtIL- 12A polypeptide (such as natural or artificial variants known in the art); and
  • a fusion protein comprising (i) a full length IL- 12A wild-type, a functional fragment or a variant thereof, and (ii) a heterologous protein.
  • the membrane domain is selected from:
  • the full-length membrane domain e.g., having the same or essentially the same length as the wild-type membrane domain
  • a functional fragment of the wild-type membrane domain e.g., a truncated (e.g., deletion of carboxy, amino terminal, or internal regions) sequence shorter than a membrane domain wild- type; but still retaining the ability of a membrane domain to tether a polypeptide to a cell membrane;
  • a variant thereof e.g., full length or truncated membrane domain proteins in which one or more amino acids have been replaced, e.g., variants that retain the ability of a membrane domain to tether a polypeptide to a cell membrane;
  • a fusion protein comprising (i) a full length membrane domain wild-type, a functional fragment or a variant thereof, and (ii) a heterologous protein.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleic acid sequence encoding a mammalian IL- 12 polypeptide and/or membrane domain (e.g., a polypeptide comprising mammalian IL- 12A, mammalian IL-12B, or both mammalian IL- 12A and mammalian IL-12B), such as a human IL- 12 polypeptide and/or membrane domain (e.g. , a polypeptide comprising human IL- 12A, human IL-12B, or both human IL- 12A and human IL- 12B), including a functional fragment or a variant thereof.
  • a mammalian IL- 12 polypeptide and/or membrane domain e.g., a polypeptide comprising mammalian IL- 12A, mammalian IL-12B, or both mammalian IL-12A and mammalian
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • IL- 12B and/or IL- 12A protein expression levels and/or IL- 12 enzymatic activity can be measured according to methods known in the art.
  • the polynucleotide is introduced to the cells in vitro. In some embodiments, the polynucleotide is introduced to the cells in vivo.
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides of the disclosure comprise a nucleotide sequence (e.g., an ORF comprising a nucleotide sequence) that encodes a wild-type human IL- 12B and/or IL- 12A.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a codon optimized nucleic acid sequence, wherein the open reading frame (ORF) of the codon optimized nucleic sequence is derived from a wild-type IL-12A and/or IL-12B sequence.
  • ORF open reading frame
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides of the disclosure comprise a nucleotide sequence encoding IL- 12B and/or IL- 12A having the full length sequence of human IL- 12B and/or IL- 12A (i.e., including the initiator methionine and the signal peptides).
  • the initiator methionine and/or signal peptides can be removed to yield a "mature IL-12B" and/or "mature IL-12A" comprising amino acid residues of SEQ ID NO: 1 and SEQ ID NO: 3, respectively.
  • SEQ ID NO: 1 corresponds to amino acids 23 to 328 of SEQ ID NO: 48
  • SEQ ID NO: 3 corresponds to amino acids 336 to 532 of SEQ ID NO: 48.
  • the teachings of the present disclosure directed to the full sequence of human IL- 12B and/or IL-12A are also applicable to the mature form of human IL- 12B and/or IL- 12A lacking the initiator methionine and/or the signal peptide.
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the disclosure comprise a nucleotide sequence encoding IL-12B and/or IL-12A having the mature sequence of human IL- 12B and/or IL- 12A.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprising a nucleotide sequence encoding IL-12B and/or IL-12A is sequence optimized.
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides of the disclosure comprise a nucleotide sequence (e.g., an ORF comprising a nucleotide sequence) encoding a tethered IL- 12 polypeptide comprising a mutant IL-12B and/or IL-12A polypeptide.
  • the polynucleotides of the disclosure comprise an ORF comprising a nucleotide sequence encoding an IL- 12B and/or IL-12A polypeptide that comprises at least one point mutation in the IL- 12B and/or IL- 12A sequence and retains IL- 12B and/or IL- 12A enzymatic activity.
  • the mutant IL-12B and/or IL- 12A polypeptide has an IL- 12B and/or IL- 12A activity which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the IL-12B and/or IL- 12A activity of the corresponding wild-type IL- 12B and/or IL- 12A (i.e., the same IL- 12B and/or IL- 12A but without the mutation(s)).
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprising an ORF encoding a mutant IL- 12B and/or IL- 12A polypeptide is sequence optimized.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleotide sequence (e.g., an ORF comprising a nucleotide sequence) that encodes an IL- 12B and/or IL- 12A polypeptide with mutations that do not alter IL- 12B and/or IL- 12A enzymatic activity.
  • a mutant IL- 12B and/or IL- 12A polypeptides can be referred to as function-neutral.
  • the polynucleotide comprises an ORF comprising a nucleotide sequence that encodes a mutant IL- 12B and/or IL- 12A polypeptide comprising one or more function-neutral point mutations.
  • the mutant IL- 12B and/or IL-12A polypeptide has higher IL- 12B and/or IL-12A enzymatic activity than the corresponding wild-type IL- 12B and/or IL- 12A.
  • the mutant IL- 12B and/or IL- 12A polypeptide has an IL- 12B and/or IL-12A activity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% higher than the activity of the corresponding wild-type IL- 12B and/or IL- 12A (i.e., the same IL- 12B and/or IL- 12A but without the mutation(s)).
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides of the disclosure comprise a nucleotide sequence (e.g., an ORF comprising a nucleotide sequence) encoding a functional IL-12B and/or IL- 12A fragment, e.g., where one or more fragments correspond to a polypeptide subsequence of a wild type IL- 12B and/or IL- 12A polypeptide and retain IL- 12B and/or IL- 12A enzymatic activity.
  • a nucleotide sequence e.g., an ORF comprising a nucleotide sequence
  • a functional IL-12B and/or IL- 12A fragment e.g., where one or more fragments correspond to a polypeptide subsequence of a wild type IL- 12B and/or IL- 12A polypeptide and retain IL-
  • the IL- 12B and/or IL- 12A fragment has an IL- 12B and/or IL-12A activity which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the IL- 12 activity of the corresponding full length IL- 12B and/or IL- 12A.
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • an ORF comprising a nucleotide sequence encoding a functional IL-12B and/or IL-12A fragment is sequence optimized.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleotide sequence (e.g., an ORF comprising a nucleotide sequence) encoding an IL- 12B and/or IL-12A fragment that has higher IL-12B and/or IL-12A enzymatic activity than the corresponding full length IL- 12B and/or IL- 12A.
  • a nucleotide sequence e.g., an ORF comprising a nucleotide sequence
  • the IL- 12B and/or IL- 12A fragment has an IL- 12B and/or IL- 12A activity which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% higher than the IL- 12B and/or IL-12A activity of the corresponding full length IL- 12B and/or IL- 12A.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleotide sequence (e.g., an ORF comprising a nucleotide sequence) encoding an IL- 12B and/or IL-12A fragment that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% shorter than wild- type IL- 12B and/or IL- 12A.
  • a nucleotide sequence e.g., an ORF comprising a nucleotide sequence
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleotide sequence (e.g., an ORF comprising a nucleotide sequence) encoding IL- 12B, which has an amino acid sequence at least about 80%, at least about 90%, at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 23 to 328 of SEQ ID NO: 48, and wherein the amino acid sequence has IL- 12B activity.
  • a nucleotide sequence e.g., an ORF comprising a nucleotide sequence
  • IL- 12B which has an amino acid sequence at least about 80%, at least about 90%, at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 23 to 328 of SEQ ID NO: 48, and wherein the amino acid sequence has IL
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleotide sequence (e.g., an ORF comprising a nucleotide sequence) encoding IL- 12A, which has an amino acid sequence at least about 80%, at least about 90%, at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 336 to 532 of SEQ ID NO:48, and wherein the amino acid sequence has IL-12A activity.
  • a nucleotide sequence e.g., an ORF comprising a nucleotide sequence
  • IL- 12A which has an amino acid sequence at least about 80%, at least about 90%, at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 336 to 532 of SEQ ID NO:48, and wherein the amino acid sequence has
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleotide sequence (e.g., an ORF comprising a nucleotide sequence) encoding IL- 12B, which has:
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleotide sequence (e.g., an ORF comprising a nucleotide sequence) encoding IL- 12A, which has:
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleotide sequence (e.g., an ORF) encoding an IL- 12B-IL- 12A fusion polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the nucleotide sequence has at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 95%, at least
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleotide sequence (e.g., an ORF comprising a nucleotide sequence) encoding an IL- 12B-IL- 12A fusion polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the nucleotide sequence has 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 70% to 95%, 80% to 95%, 70% to 85%, 75% to 90%, 80% to 95%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100%, sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 5 to 44 or 220.
  • a nucleotide sequence e.g., an ORF comprising a nucleotide sequence
  • the polynucleotide encoding the IL-12 polypeptide comprises a nucleotide sequence at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6 (hIL12AB_002).
  • the polynucleotide encoding the IL-12 polypeptide comprises a nucleotide sequence at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 220 (hIL12AB_041).
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises from about 900 to about 100,000 nucleotides (e.g., from 900 to 1,000, from 900 to 1,100, from 900 to 1,200, from 900 to 1,300, from 900 to 1,400, from 900 to 1,500, from 1,000 to 1,100, from 1,000 to 1,100, from 1,000 to 1,200, from 1,000 to 1,300, from 1,000 to 1,400, from 1,000 to 1,500, from 1,083 to 1,200, from 1,083 to 1,400, from 1,083 to 1,600, from 1,083 to 1,800, from 1,083 to 2,000, from 1,083 to 3,000, from 1,083 to 5,000, from 1,083 to 7,000, from 1,083 to 10,000, from 1,083 to 25,000, from 1,083 to 50,000, from 1,083 to 70,000, or from 1,083 to 100,000).
  • nucleotides e.g., from 900 to 1,000, from 900
  • the polynucleotide of the disclosure (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence encoding an IL- 12B-IL- 12A fusion polypeptide (e.g., an ORF comprising a nucleotide sequence encoding IL- 12B and a nucleotide encoding IL-12A; e.g.
  • nucleic acid sequence encoding the IL-12B and the nucleic acid sequence encoding the IL-12A are fused directly to one another.
  • nucleic acid sequence encoding the IL- 12B and the nucleic acid sequence (encoding the IL- 12A are linked by a nucleic acid sequence encoding a subunit linker.
  • the nucleic acid sequence encoding the membrane domain is fused directly to the nucleic acid sequence encoding the IL- 12 polypeptide comprising IL-12A, IL-12B, or IL- 12A and IL-12B.
  • the nucleic acid sequence encoding the membrane domain is linked to the nucleic acid sequence encoding the IL- 12 polypeptide comprising IL- 12A, IL- 12B, or IL-12A and IL-12B by a nucleic acid sequence encoding a membrane domain linker.
  • the polynucleotide of the disclosure (e.g., a RNA, e.g., an mRNA) further comprises at least one nucleic acid sequence that is noncoding, e.g., a miRNA binding site.
  • the polynucleotide of the disclosure e.g., a RNA, e.g., an mRNA
  • a RNA e.g., an mRNA
  • the polynucleotide of the disclosure is single stranded or double stranded.
  • the polynucleotide of the disclosure is DNA or RNA. In some embodiments, the polynucleotide of the disclosure is RNA. In some embodiments, the
  • polynucleotide of the disclosure is, or functions as, a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the mRNA comprises a nucleotide sequence that encodes IL- 12B and/or IL- 12A and a nucleic acid sequence encoding a membrane domain, and is capable of being translated to produce the encoded tethered IL- 12 polypeptide in vitro, in vivo, in situ or ex vivo.
  • the polynucleotide of the disclosure (e.g., a RNA, e.g., an mRNA) comprises a sequence-optimized nucleotide sequence encoding IL- 12B and/or IL- 12A (e.g., the wild-type sequence, functional fragment, or variant thereof encoding the IL-12B and/or the IL-12 A) and/or a sequence-optimized nucleotide sequence encoding the membrane domain, wherein the polynucleotide comprises at least one chemically modified nucleobase, e.g., 5-methoxyuracil.
  • a sequence-optimized nucleotide sequence encoding IL- 12B and/or IL- 12A
  • the polynucleotide comprises at least one chemically modified nucleobase, e.g., 5-methoxyuracil.
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miR- 122.
  • the polynucleotide disclosed herein is formulated with a delivery agent, e.g., a compound having Formula (I).
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • One such feature that aids in protein trafficking is the signal sequence, or targeting sequence.
  • the peptides encoded by these signal sequences are known by a variety of names, including targeting peptides, transit peptides, and signal peptides.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF comprising a nucleotide sequence) that encodes a signal peptide operably linked a nucleotide sequence that encodes an IL-12B and/or IL-12A polypeptide described herein.
  • a nucleotide sequence e.g., an ORF comprising a nucleotide sequence
  • a signal peptide operably linked a nucleotide sequence that encodes an IL-12B and/or IL-12A polypeptide described herein.
  • the "signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3-70 amino acids) in length that, optionally, is incorporated at the 5' (or N-terminus) of the coding region or the polypeptide, respectively.
  • Some signal peptides are cleaved from the protein, for example by a signal peptidase after the proteins are transported to the desired site.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleotide sequence encoding an IL- 12B and/or IL- 12A polypeptide, wherein the nucleotide sequence further comprises a 5' nucleic acid sequence encoding a native signal peptide.
  • the polynucleotide of the disclosure comprises a nucleotide sequence encoding an IL- 12B and/or IL- 12A polypeptide, wherein the nucleotide sequence lacks the nucleic acid sequence encoding a native signal peptide.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleotide sequence encoding an IL- 12B and/or IL- 12A polypeptide, wherein the nucleotide sequence further comprises a 5' nucleic acid sequence encoding a heterologous signal peptide.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) further comprises a nucleic acid sequence encoding a signal peptide that is located at the 5' terminus of the nucleotide sequence encoding the IL-12B.
  • the nucleotide sequence encoding the IL-12B comprises a nucleic acid sequence encoding a signal peptide.
  • the signal peptide comprises a sequence at least about 80%, at least about 90%, at least about 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 1 to 22 of SEQ ID NO: 48.
  • the disclosure provides mRNA comprising an open reading frame (ORF), wherein the ORF comprises a nucleotide sequence encoding from 5' to 3' :
  • IL- 12B is a human IL- 12 p40 subunit polypeptide
  • LI is a first peptide linker
  • IL- 12A is a human IL- 12 p35 subunit polypeptide
  • L2 is a second peptide linker
  • MD is a membrane domain comprising a transmembrane domain, and optionally an intracellular domain.
  • the polynucleotide (e.g., mRNA) encoding a tethered IL- 12 polypeptide comprises an open reading frame (ORF) encoding IL- 12B, IL- 12A, and a CD8 transmembrane domain, wherein the ORF optionally encodes a linker that links the IL-12B and the IL-12A, and wherein the ORF optionally encodes a linker that links the CD8 transmembrane domain and the IL- 12B.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding IL- 12B, IL- 12A, and a CD8 transmembrane domain, wherein the ORF optionally encodes a linker that links the IL- 12B and the IL- 12A, and wherein the ORF optionally encodes a linker that links the CD8 transmembrane domain and the IL-12A.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding IL- 12B, IL- 12A, and a CD80 transmembrane domain, wherein the ORF optionally encodes a linker that links the IL- 12B and the IL- 12A, and wherein the ORF optionally encodes a linker that links the CD80 transmembrane domain and the IL- 12B.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding IL- 12B, IL- 12A, and a CD80 transmembrane domain, wherein the ORF optionally encodes a linker that links the IL- 12B and the IL- 12A, and wherein the ORF optionally encodes a linker that links the CD80 transmembrane domain and the IL- 12A.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding IL-12B, IL-12 A, and a PDGFR-beta transmembrane domain, wherein the ORF optionally encodes a linker that links the IL- 12B and the IL- 12A, and wherein the ORF optionally encodes a linker that links the PDGFR-beta transmembrane domain and the IL- 12B.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding IL-12B, IL-12 A, and a PDGFR-beta transmembrane domain, wherein the ORF optionally encodes a linker that links the IL- 12B and the IL- 12A, and wherein the ORF optionally encodes a linker that links the PDGFR-beta transmembrane domain and the IL- 12A.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding IL- 12B, IL- 12A, a CD80 transmembrane domain and intracellular domain, wherein the ORF optionally encodes a linker that links the IL-12B and the IL-12A, and wherein the ORF optionally encodes a linker that links the CD80 transmembrane domain and the IL-12B.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding IL- 12B, IL- 12A, a CD80 transmembrane domain and intracellular domain, wherein the ORF optionally encodes a linker that links the IL-12B and the IL-12A, and wherein the ORF optionally encodes a linker that links the CD80 transmembrane domain and the IL-12A.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding IL- 12B, IL- 12A, a PDGFR-beta transmembrane domain and intracellular domain, wherein the ORF optionally encodes a linker that links the IL-12B and the IL-12A, and wherein the ORF optionally encodes a linker that links the CD80 transmembrane domain and the IL-12B.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding IL- 12B, IL- 12A, a PDGFR-beta transmembrane domain and intracellular domain, wherein the ORF optionally encodes a linker that links the IL-12B and the IL-12A, and wherein the ORF optionally encodes a linker that links the CD80 transmembrane domain and the IL-12A.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding IL- 12B, IL- 12A, a PDGFR-beta transmembrane domain and truncated intracellular domain, wherein the ORF optionally encodes a linker that links the IL- 12B and the IL- 12A, and wherein the ORF optionally encodes a linker that links the CD80 transmembrane domain and the IL- 12B.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding IL- 12B, IL- 12A, a PDGFR-beta transmembrane domain and truncated intracellular domain, wherein the ORF optionally encodes a linker that links the IL- 12B and the IL- 12A, and wherein the ORF optionally encodes a linker that links the CD80 transmembrane domain and the IL- 12A.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding an IL-12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 48, a linker comprising the amino acid sequence set forth in SEQ ID NO: 229, and a CD8 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 101.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding an IL-12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 48, a linker comprising the amino acid sequence set forth in SEQ ID NO: 229, and a CD80 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 103.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding an IL-12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 48, a linker comprising the amino acid sequence set forth in SEQ ID NO: 229, and a PDGFR-beta transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 102.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding an IL-12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 48, a linker comprising the amino acid sequence set forth in SEQ ID NO: 229, a CD80 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 103, and a CD80 intracellular domain comprising the amino acid sequence set forth in SEQ ID NO: 225.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding an IL-12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 48, a linker comprising the amino acid sequence set forth in SEQ ID NO: 229, a CD80 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 103, and a truncated PDGFR-beta intracellular domain comprising the amino acid sequence set forth in SEQ ID NO: 227.
  • ORF open reading frame
  • the polynucleotide (e.g., mRNA) encoding a tethered IL-12 polypeptide comprises an open reading frame (ORF) encoding an IL-12 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 48, a linker comprising the amino acid sequence set forth in SEQ ID NO: 229, a CD80 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 103, and a truncated PDGFR-beta intracellular domain comprising the amino acid sequence set forth in SEQ ID NO: 228.
  • ORF open reading frame
  • the polynucleotide encoding a tethered IL-12 polypeptide is an mRNA comprising an open reading frame (ORF), comprising SEQ ID NO: 221, SEQ ID NO: 376, and SEQ ID NO: 377.
  • the polynucleotide encoding a tethered IL-12 polypeptide is an mRNA comprising a 5'UTR comprising SEQ ID NO: 287; an open reading frame (ORF), comprising SEQ ID NO: 221, SEQ ID NO: 376, and SEQ ID NO: 377; and a 3'UTR comprising SEQ ID NO: 283.
  • the polynucleotide encoding a tethered IL-12 polypeptide is an mRNA comprising an open reading frame (ORF) comprising SEQ ID NO: 221, SEQ ID NO: 376, and SEQ ID NO: 378.
  • the polynucleotide encoding a tethered IL-12 polypeptide is an mRNA comprising a 5'UTR comprising SEQ ID NO: 287; an open reading frame (ORF) comprising SEQ ID NO: 221, SEQ ID NO: 376, and SEQ ID NO: 378; and a 3'UTR comprising SEQ ID NO: 283.
  • the polynucleotide encoding a tethered IL-12 polypeptide is an mRNA comprising an open reading frame (ORF) comprising SEQ ID NO: 221, SEQ ID NO: 376, and SEQ ID NO: 250.
  • the polynucleotide encoding a tethered IL-12 polypeptide is an mRNA comprising a 5'UTR comprising SEQ ID NO: 287; an open reading frame (ORF) comprising SEQ ID NO: 221, SEQ ID NO: 376, and SEQ ID NO: 250; and a 3'UTR comprising SEQ ID NO: 283.
  • the polynucleotide encoding a tethered IL-12 polypeptide is an mRNA comprising an open reading frame (ORF) comprising SEQ ID NO: 221, SEQ ID NO: 376, SEQ ID NO: 378 and SEQ ID NO: 379.
  • ORF open reading frame
  • the polynucleotide encoding a tethered IL-12 polypeptide is an mRNA comprising a 5'UTR comprising SEQ ID NO: 287; an open reading frame (ORF) comprising SEQ ID NO: 221, SEQ ID NO: 376, SEQ ID NO: 378 and SEQ ID NO: 379; and a 3'UTR comprising SEQ ID NO: 283.
  • the polynucleotide encoding a tethered IL-12 polypeptide is an mRNA comprising an open reading frame (ORF) comprising SEQ ID NO: 221, SEQ ID NO: 376, SEQ ID NO: 250 and SEQ ID NO: 251.
  • the polynucleotide encoding a tethered IL-12 polypeptide is an mRNA comprising a 5'UTR comprising SEQ ID NO: 287; an open reading frame (ORF) comprising SEQ ID NO: 221, SEQ ID NO: 376, SEQ ID NO: 250 and SEQ ID NO: 325; and a 3'UTR comprising SEQ ID NO: 283.
  • the polynucleotide encoding a tethered IL- 12 polypeptide is an mRNA comprising an open reading frame (ORF) comprising SEQ ID NO: 221, SEQ ID NO: 376, SEQ ID NO: 250 and SEQ ID NO: 280.
  • the polynucleotide encoding a tethered IL- 12 polypeptide is an mRNA comprising a 5'UTR comprising SEQ ID NO: 287; an open reading frame (ORF) comprising SEQ ID NO: 221, SEQ ID NO: 376, SEQ ID NO: 250 and SEQ ID NO: 280; and a 3 'UTR comprising SEQ ID NO: 283.
  • the polynucleotide of the disclosure can comprise more than one nucleic acid sequence encoding one or more polypeptides.
  • polynucleotides of the disclosure comprise an a nucleic acid sequence encoding an IL- 12 polypeptide comprising IL- 12A and/or IL-12B and a nucleic acid sequence encoding a membrane domain, including functional fragments or a variants thereof (i.e. , any or all of the IL- 12A, IL- 12B, or the membrane domain can be a functional fragment or variant).
  • the polynucleotide of the disclosure can comprise a nucleic acid sequence encoding IL-12B, a nucleic acid sequence encoding IL-12 A, and a nucleic acid sequence encoding a membrane domain, including functional fragments or a variant thereof (i.e. , any or all of the IL-12A, IL- 12B, or the membrane domain can be a functional fragment or variant).
  • the membrane domain including functional fragments or a variant thereof (i.e. , any or all of the IL-12A, IL- 12B, or the membrane domain can be a functional fragment or variant).
  • the membrane domain including functional fragments or a variant thereof (i.e. , any or all of the IL-12A, IL- 12B, or the membrane domain can be a functional fragment or variant).
  • the membrane domain including functional fragments or a variant thereof (i.e. , any or all of the IL-12A, IL- 12B, or the membrane domain can be a functional fragment or variant).
  • polynucleotide of the disclosure can comprise one or more additional nucleic acid sequences expressing one or more additional polypeptides of interest (e.g., one or more additional nucleic acid sequences encoding one or more polypeptides heterologous to IL-12).
  • the additional polypeptide of interest can be fused to the IL-12B polypeptide directly or by a linker.
  • the additional polypeptide of interest can be fused to the IL-12A polypeptide directly or by a linker.
  • the additional polypeptide of interest can be fused to both the IL- 12B polypeptide and the IL-12A polypeptide directly or by a linker.
  • the first additional polypeptide of interest is fused to the IL- 12A polypeptide directly or by a linker
  • the second additional polypeptide of interest is fused to the IL-12B polypeptide directly or by a linker.
  • two or more additional polypeptides of interest can be genetically fused, i.e., two or more additional polypeptides of interest can be encoded by the same ORF.
  • the polynucleotide can comprise a nucleic acid sequence encoding a linker (e.g., a G 4 S peptide linker or another linker known in the art) between two or more additional polypeptides of interest.
  • the polynucleotide of the disclosure can comprise a nucleic acid sequence (e.g., one or more nucleic acid sequences) encoding an IL- 12B polypeptide, IL- 12A polypeptide, both IL- 12B and IL- 12A polypeptides, a nucleic acid sequence encoding a membrane domain, and one or more additional nucleic acid sequences encoding one or more additional polypeptides of interest.
  • a nucleic acid sequence e.g., one or more nucleic acid sequences
  • the membrane domain can be fused directly to the IL-12 polypeptide of the present disclosure comprising IL- 12A, IL- 12B, and/or IL- 12A and IL-12B or can be linked to the IL- 12 polypeptide by a linker (referred to herein as the "membrane domain linker" or a
  • IL-12B and IL- 12A in an IL- 12 polypeptide can be fused directly to one another or can be linked to one another by a linker (referred to herein as the "subunit linker”).
  • the IL- 12B and/or IL-12A can be fused directly to a heterologous polypeptide or can be linked to the heterologous polypeptide by a linker (referred to herein as the "heterologous polypeptide linker.”).
  • the membrane domain can be fused directly to a heterologous polypeptide or can be linked to the heterologous polypeptide by heterologous polypeptide linker.
  • Suitable linkers can be a polypeptide (or peptide) moiety or a non-polypeptide moiety.
  • the linker is a peptide linker, including from one amino acid to about 200 amino acids.
  • the linker comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 amino acids.
  • linker-encoding sequences (in particular, flexible linker-encoding sequences) between sequences (e.g. , ORF sequences) encoding functional domains (e.g. , interleukin chains, transmembrane domains, etc.) provide a certain degree of movement or interaction between domains, thus improving functionality of the mRNA-encoded chimeric (e.g. , "tethered") interleukins of the disclosure.
  • Flexible linkers are generally composed of small, non-polar (e.g. , Gly) or polar (e.g. , Ser) amino acids. The small size of these amino acids provides flexibility, and allows for mobility of the connected functional domains.
  • Preferred flexible linkers encoded by sequences within the mRNAs of the disclosure have sequences consisting primarily of stretches of Gly and Ser residues ("GS" linker).
  • An exemplary flexible linker has the sequence of (Gly-Gly-Gly-Gly-Ser) n .
  • the length of this GS linker can be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions.
  • the membrane domain linker is of sufficient length to prevent steric hindrance from the cell membrane.
  • the linker can be a GS (Gly/Ser) linker, for example, comprising (GnS)m, wherein n is an integer from 1 to 100 and m is an integer from 1 to 100.
  • the Gly/Ser linker comprises (G n S) m (SEQ ID NO: 193), wherein n is from 1 to 20, e.g., 1, 2 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 and m is from 1 to 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20.
  • the GS linker can comprise (GGGGS) 0 (SEQ ID NO: 194), wherein o is an integer from 1 to 5.
  • the GS linker can comprise GGSGGGGSGG (SEQ ID NO: 195), GGSGGGGG (SEQ ID NO: 196), or GSGSGSGS (SEQ ID NO: 197). In some embodiments, the GS linker comprises GGGGGGS (SEQ ID NO: 214).
  • the linker suitable for the disclosure can be a Gly-rich linker, for example, comprising (Gly) p (SEQ ID NO: 198), wherein p is an integer from 1 to 100, e.g., from 1 to 40.
  • a Gly-rich linker can comprise GGGGG (SEQ ID NO: 192), GGGGGG (SEQ ID NO: 217), GGGGGGG (SEQ ID NO: 218) or GGGGGGGG (SEQ ID NO: 219).
  • the linker suitable for the disclosure can comprise (EAAAK) q (SEQ ID NO: 199), wherein q is an integer from 1 to 100, e.g., from 1 to 20, e.g., from 1 to 5. In one embodiment, the linker suitable for the disclosure can comprise (EAAAK) 3 .
  • linkers include, but not limited to, GGGGS LVPRGS GGGGS (SEQ ID NO: 200), GSGSGS (SEQ ID NO: 201), GGGGS LVPRGS GGGG (SEQ ID NO: 202), GGSGGHMGSGG (SEQ ID NO: 203), GGSGGSGGSGG (SEQ ID NO: 204), GGSGG (SEQ ID NO: 205), GSGSGSGS (SEQ ID NO: 206), GGGSEGGGSEGGGSEGGG (SEQ ID NO: 207), AAGAATAA (SEQ ID NO: 208), GGSSG (SEQ ID NO: 209), GS GGGTGGGS G (SEQ ID NO: 210), GSGSGSGSGSGGSG (SEQ ID NO: 211), GSGGSGSGGSGGSG (SEQ ID NO: 212), and GSGGSGGSGGSGGS (SEQ ID NO: 213).
  • the nucleotides encoding the linkers can be constructed to fuse the sequences of the present disclosure. Based on the RNA sequences provided, a person of ordinary skill in the art would understand the corresponding DNA sequence (e.g., conversion of uracil to thymine). Likewise, based on the DNA sequences provided, a person of ordinary skill in the art would understand the corresponding RNA sequence (e.g., conversion of thymine to uracil).
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure is sequence optimized.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a nucleotide sequence encoding an IL- 12 polypeptide (i.e.
  • an IL- 12B and/or IL- 12A polypeptide a nucleotide sequence encoding a membrane domain, a nucleotide sequence encoding another polypeptide of interest, a 5'-UTR, a 3'-UTR, a miRNA, a nucleotide sequence encoding a linker (e.g. , a membrane domain linker, a subunit linker, and/or a heterologous polypeptide linker), or any combination thereof that is sequence optimized.
  • a linker e.g. , a membrane domain linker, a subunit linker, and/or a heterologous polypeptide linker
  • a sequence-optimized nucleotide sequence (e.g., an codon-optimized mRNA sequence encoding an IL- 12B and/or IL- 12A polypeptide) is a sequence comprising at least one synonymous nucleobase substitution with respect to a reference sequence (e.g., a wild type nucleotide sequence encoding an IL- 12B and/or IL- 12A polypeptide).
  • a sequence-optimized nucleotide sequence can be partially or completely different in sequence from the reference sequence.
  • a reference sequence encoding polyserine uniformly encoded by TCT codons can be sequence-optimized by having 100% of its nucleobases substituted (for each codon, T in position 1 replaced by A, C in position 2 replaced by G, and T in position 3 replaced by C) to yield a sequence encoding polyserine which would be uniformly encoded by AGC codons.
  • the percentage of sequence identity obtained from a global pairwise alignment between the reference polyserine nucleic acid sequence and the sequence-optimized polyserine nucleic acid sequence would be 0%.
  • the protein products from both sequences would be 100% identical.
  • sequence optimization also sometimes referred to codon optimization
  • results can include, e.g., matching codon frequencies in certain tissue targets and/or host organisms to ensure proper folding; biasing G/C content to increase mRNA stability or reduce secondary structures; minimizing tandem repeat codons or base runs that can impair gene construction or expression; customizing transcriptional and translational control regions; inserting or removing protein trafficking sequences; removing/adding post translation modification sites in an encoded protein (e.g.
  • sequence optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. Codon options for each amino acid are given in Table 1.
  • a polynucleotide (e.g., a RNA, e.g., an mRNA) of the disclosure comprises a sequence-optimized nucleotide sequence encoding an IL-12 polypeptide, including a functional fragment or a variant thereof, or a sequence-optimized nucleotide sequence encoding a membrane domain, including a functional fragment or a variant thereof, wherein the IL- 12 polypeptide and/or membrane domain encoded by the sequence-optimized nucleotide sequence has improved properties (e.g., compared to an IL-12 polypeptide, including a functional fragment or a variant thereof, or a membrane domain, including a functional fragment or a variant thereof, encoded by a reference nucleotide sequence that is not sequence optimized), e.g., improved properties related to expression efficacy after administration in vivo.
  • Such properties include, but are not limited to, improving nucleic acid stability (e.g., mRNA stability), increasing translation efficacy in the target tissue, reducing the number of truncated proteins expressed, improving the folding or prevent misfolding of the expressed proteins, reducing toxicity of the expressed products, reducing cell death caused by the expressed products, increasing and/or decreasing protein aggregation.
  • nucleic acid stability e.g., mRNA stability
  • increasing translation efficacy in the target tissue reducing the number of truncated proteins expressed, improving the folding or prevent misfolding of the expressed proteins, reducing toxicity of the expressed products, reducing cell death caused by the expressed products, increasing and/or decreasing protein aggregation.
  • the sequence-optimized nucleotide sequence is codon optimized for expression in human subjects, having structural and/or chemical features that avoid one or more of the problems in the art, for example, features which are useful for optimizing formulation and delivery of nucleic acid-based therapeutics while retaining structural and functional integrity; overcoming a threshold of expression; improving expression rates; half-life and/or protein concentrations; optimizing protein localization; and avoiding deleterious bio-responses such as the immune response and/or degradation pathways.
  • the polynucleotides of the disclosure comprise a nucleotide sequence (e.g., a nucleotide sequence encoding an IL-12 polypeptide, a nucleotide sequence encoding a membrane domain, a nucleotide sequence encoding an additional polypeptide of interest, a 5'-UTR, a 3'-UTR, a microRNA, a nucleic acid sequence encoding a linker, or any combination thereof) that is sequence-optimized according to a method comprising:
  • a reference nucleotide sequence e.g., a nucleic acid sequence encoding an IL- 12 polypeptide and/or a nucleic acid sequence encoding a membrane domain
  • the sequence-optimized nucleotide sequence (e.g., a nucleic acid sequence encoding an IL- 12 polypeptide and/or a nucleic acid sequence encoding a membrane domain) has at least one improved property with respect to the reference nucleotide sequence.
  • the sequence optimization method is multiparametric and comprises one, two, three, four, or more methods disclosed herein and/or other optimization methods known in the art.
  • regions of the polynucleotide can be encoded by or within regions of the polynucleotide and such regions can be upstream (5') to, downstream (3') to, or within the region that encodes the IL- 12 polypeptide and/or the region that encodes membrane domain. These regions can be incorporated into the polynucleotide before and/or after sequence-optimization of the protein encoding region or open reading frame (ORF). Examples of such features include, but are not limited to, untranslated regions (UTRs), microRNA sequences, Kozak sequences, oligo(dT) sequences, poly- A tail, and detectable tags and can include multiple cloning sites that may have Xbal recognition.
  • the polynucleotide of the disclosure comprises a 5' UTR, a 3 ' UTR and/or a miRNA. In some embodiments, the polynucleotide comprises two or more 5' UTRs and/or 3 ' UTRs, which can be the same or different sequences. In some embodiments, the polynucleotide comprises two or more miRNA, which can be the same or different sequences. Any portion of the 5' UTR, 3' UTR, and/or miRNA, including none, can be sequence-optimized and can independently contain one or more different structural or chemical modifications, before and/or after sequence optimization.
  • the polynucleotide is reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
  • a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
  • the optimized polynucleotide can be reconstituted and transformed into chemically competent E. coli, yeast, neurospora, maize, drosophila, etc. where high copy plasmid-like or chromosome structures occur by methods described herein. 9. Sequence-Optimized Nucleotide Sequences Encoding IL-12 Polypeptides
  • the polynucleotide of the disclosure comprises a sequence-optimized nucleotide sequence encoding an IL- 12 polypeptide (i.e. , an IL- 12B and/or IL- 12A polypeptide) disclosed herein.
  • the polynucleotide of the disclosure comprises a nucleic acid sequence encoding an IL- 12B and/or a nucleic acid sequence encoding an IL- 12A polypeptide and a nucleic acid sequence encoding a membrane domain, wherein the nucleic acid sequences have been sequence optimized.
  • Exemplary sequence-optimized nucleotide sequences encoding human IL- 12B and/or IL- 12A are set forth in SEQ ID NOs: 5-44, 98- 100, 104-180, 220 and 221.
  • sequence optimized IL-12B and/or IL-12A sequences set forth in SEQ ID NOs: 5-44, 98- 100, 104- 180, 220 and 221, fragments, and variants thereof are used to practice the methods disclosed herein.
  • sequence optimized IL- 12B and/or IL- 12A sequences set forth in SEQ ID NOs: 5-44, 98- 100, 104- 180, 220 and 221, fragments and variants thereof are combined with or alternatives to the wild-type sequences disclosed in SEQ ID NOs: 1-6.
  • RNA sequences provided a person of ordinary skill in the art would understand the corresponding DNA sequence (e.g., conversion of uracil to thymine).
  • corresponding RNA sequence e.g., conversion of thymine to uracil.
  • sequence-optimized nucleotide sequences disclosed herein are distinct from the corresponding wild type nucleotide acid sequences and from other known sequence-optimized nucleotide sequences, e.g., these sequence-optimized nucleic acids have unique compositional characteristics.
  • a polynucleotide of the disclosure e.g., a polynucleotide comprising a nucleotide sequence encoding an IL- 12 polypeptide comprising IL- 12A, IL- 12B, or both IL- 12A and IL- 12B (e.g., the wild-type sequence, functional fragment, or variant thereof) and a membrane domain
  • a polynucleotide of the disclosure is sequence optimized.
  • a sequence optimized nucleotide sequence (nucleotide sequence is also referred to as "nucleic acid" herein) comprises at least one codon modification with respect to a reference sequence (e.g., a wild-type sequence encoding an IL-12 polypeptide comprising IL- 12A, IL- 12B, or both IL- 12A and IL- 12B or a membrane domain).
  • a reference sequence e.g., a wild-type sequence encoding an IL-12 polypeptide comprising IL- 12A, IL- 12B, or both IL- 12A and IL- 12B or a membrane domain.
  • a reference sequence e.g., a wild- type sequence
  • sequence optimized nucleic acids are generated by at least a step comprising substituting codons in a reference sequence with synonymous codons (i.e., codons that encode the same amino acid).
  • substitutions can be effected, for example, by applying a codon substitution map (i.e., a table providing the codons that will encode each amino acid in the codon optimized sequence), or by applying a set of rules (e.g., if glycine is next to neutral amino acid, glycine would be encoded by a certain codon, but if it is next to a polar amino acid, it would be encoded by another codon).
  • sequence optimization methods disclosed herein comprise additional optimization steps which are not strictly directed to codon optimization such as the removal of deleterious motifs (destabilizing motif substitution).
  • compositions and formulations comprising these sequence optimized nucleic acids (e.g., a RNA, e.g., an mRNA) can be administered to a subject in need thereof to facilitate in vivo expression of functionally active IL- 12.
  • sequence optimized nucleic acids e.g., a RNA, e.g., an mRNA
  • RNA e.g., an mRNA
  • IL- 12 a functionally active IL- 12 or compositions or formulations comprising the same
  • Changing from an in vitro expression system (e.g., cell culture) to in vivo expression requires the redesign of the nucleic acid sequence encoding the therapeutic agent.
  • Redesigning a naturally occurring gene sequence by choosing different codons without necessarily altering the encoded amino acid sequence can often lead to dramatic increases in protein expression levels (Gustafsson et al., 2004, Journal/Trends Biotechnol 22, 346-53).
  • Variables such as codon adaptation index (CAI), mRNA secondary structures, cis-regulatory sequences, GC content and many other similar variables have been shown to somewhat correlate with protein expression levels (Villalobos et al., 2006, "Journal/BMC Bioinformatics 7, 285).
  • codon usage i.e., the frequency with which different organisms use codons for expressing a polypeptide sequence
  • codon usage differs among organisms (for example, recombinant production of human or humanized therapeutic antibodies frequently takes place in hamster cell cultures).
  • a reference nucleic acid sequence can be sequence optimized by applying a codon map.
  • T bases in the codon maps disclosed below are present in DNA, whereas the T bases would be replaced by U bases in corresponding RNAs.
  • a sequence optimized nucleic acid disclosed herein in DNA form e.g., a vector or an in-vitro translation (IVT) template, would have its T bases transcribed as U based in its corresponding transcribed mRNA.
  • IVTT in-vitro translation
  • a TTC codon (DNA map) would correspond to a UUC codon (RNA map), which in turn may correspond to a ⁇ codon (RNA map in which U has been replaced with pseudouridine).
  • a reference sequence encoding IL- 12A, IL- 12B, or both IL- 12A and IL- 12B and/or a membrane domain can be optimized by replacing all the codons encoding a certain amino acid with only one of the alternative codons provided in a codon map.
  • all the valines in the optimized sequence would be encoded by GTG or GTC or GTT.
  • Sequence optimized polynucleotides of the disclosure can be generated using one or more codon optimization methods, or a combination thereof. Sequence optimization methods which may be used to sequence optimize nucleic acid sequences are described in detail herein. This list of methods is not comprehensive or limiting.
  • sequence optimization methods can be, for example, dependent on the specific chemistry used to produce a synthetic polynucleotide. Such a choice can also depend on characteristics of the protein encoded by the sequence optimized nucleic acid, e.g., a full sequence, a functional fragment, or a fusion protein comprising IL-12, etc. In some embodiments, such a choice can depend on the specific tissue or cell targeted by the sequence optimized nucleic acid (e.g., a therapeutic synthetic mRNA).
  • the mechanisms of combining the sequence optimization methods or design rules derived from the application and analysis of the optimization methods can be either simple or complex.
  • the combination can be:
  • Sequential Each sequence optimization method or set of design rules applies to a different subsequence of the overall sequence, for example reducing uridine at codon positions 1 to 30 and then selecting high frequency codons for the remainder of the sequence;
  • Hierarchical Several sequence optimization methods or sets of design rules are combined in a hierarchical, deterministic fashion. For example, use the most GC-rich codons, breaking ties (which are common) by choosing the most frequent of those codons.
  • Multifactorial / Multiparametric Machine learning or other modeling techniques are used to design a single sequence that best satisfies multiple overlapping and possibly contradictory requirements. This approach would require the use of a computer applying a number of mathematical techniques, for example, genetic algorithms.
  • each one of these approaches can result in a specific set of rules which in many cases can be summarized in a single codon table, i.e., a sorted list of codons for each amino acid in the target protein (i.e., an IL-12A polypeptide, an IL-12B polypeptide, both IL-12A and IL-12B polypeptides, and/or a membrane domain), with a specific rule or set of rules indicating how to select a specific codon for each amino acid position.
  • a nucleic acid sequence disclosed herein e.g., a nucleic acid sequence encoding an IL-12 polypeptide and/or a nucleic acid sequence encoding a membrane domain
  • a nucleic acid sequence disclosed herein can be sequence optimized using methods comprising the use of modifications in the frequency of use of one or more codons relative to other synonymous codons in the sequence optimized nucleic acid with respect to the frequency of use in the non-codon optimized sequence.
  • codon usage bias i.e., the differences in the frequency of occurrence of synonymous codons in coding DNA/RNA. It is generally acknowledged that codon preferences reflect a balance between mutational biases and natural selection for translational optimization. Optimal codons help to achieve faster translation rates and high accuracy. As a result of these factors, translational selection is expected to be stronger in highly expressed genes. In the field of bioinformatics and computational biology, many statistical methods have been proposed and used to analyze codon usage bias. See, e.g., Comeron & Aguade (1998) J. Mol. Evol. 47: 268-74.
  • Multivariate statistical methods such as correspondence analysis and principal component analysis, are widely used to analyze variations in codon usage among genes (Suzuki et al. (2008) DNA Res. 15 (6): 357-65; Sandhu et al., In Silico Biol. 2008;8(2): 187-92).
  • a nucleic acid sequence disclosed herein can be codon optimized using methods comprising substituting at least one codon in the reference nucleic acid sequence with an alternative codon having a higher or lower codon frequency in the synonymous codon set; wherein the resulting sequence optimized nucleic acid has at least one optimized property with respect to the reference nucleic acid sequence.
  • At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% of the codons in the reference nucleic acid sequence are substituted with alternative codons, each alternative codon having a codon frequency higher than the codon frequency of the substituted codon in the synonymous codon set.
  • At least one codon in the reference nucleic acid sequence is substituted with an alternative codon having a codon frequency higher than the codon frequency of the substituted codon in the synonymous codon set, and at least one codon in the reference nucleic acid sequence is substituted with an alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.
  • At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% of the codons in the reference nucleic acid sequence are substituted with alternative codons, each alternative codon having a codon frequency higher than the codon frequency of the substituted codon in the synonymous codon set.
  • At least one alternative codon having a higher codon frequency has the highest codon frequency in the synonymous codon set. In other embodiments, all alternative codons having a higher codon frequency have the highest codon frequency in the synonymous codon set.
  • At least one alternative codon having a lower codon frequency has the lowest codon frequency in the synonymous codon set. In some embodiments, all alternative codons having a higher codon frequency have the highest codon frequency in the synonymous codon set.
  • At least one alternative codon has the second highest, the third highest, the fourth highest, the fifth highest or the sixth highest frequency in the synonymous codon set. In some specific embodiments, at least one alternative codon has the second lowest, the third lowest, the fourth lowest, the fifth lowest, or the sixth lowest frequency in the synonymous codon set.
  • Optimization based on codon frequency can be applied globally, as described above, or locally to the reference nucleic acid sequence.
  • regions of the reference nucleic acid sequence can modified based on codon frequency, substituting all or a certain percentage of codons in a certain subsequence with codons that have higher or lower frequencies in their respective synonymous codon sets.
  • At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% of the codons in a subsequence of the reference nucleic acid sequence are substituted with alternative codons, each alternative codon having a codon frequency higher than the codon frequency of the substituted codon in the synonymous codon set.
  • At least one codon in a subsequence of the reference nucleic acid sequence is substituted with an alternative codon having a codon frequency higher than the codon frequency of the substituted codon in the synonymous codon set, and at least one codon in a subsequence of the reference nucleic acid sequence is substituted with an alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.
  • At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% of the codons in a subsequence of the reference nucleic acid sequence are substituted with alternative codons, each alternative codon having a codon frequency higher than the codon frequency of the substituted codon in the synonymous codon set.
  • At least one alternative codon substituted in a subsequence of the reference nucleic acid sequence and having a higher codon frequency has the highest codon frequency in the synonymous codon set. In other embodiments, all alternative codons substituted in a subsequence of the reference nucleic acid sequence and having a lower codon frequency have the lowest codon frequency in the synonymous codon set.
  • At least one alternative codon substituted in a subsequence of the reference nucleic acid sequence and having a lower codon frequency has the lowest codon frequency in the synonymous codon set. In some embodiments, all alternative codons substituted in a subsequence of the reference nucleic acid sequence and having a higher codon frequency have the highest codon frequency in the synonymous codon set.
  • a sequence optimized nucleic acid can comprise a subsequence having an overall codon frequency higher or lower than the overall codon frequency in the corresponding subsequence of the reference nucleic acid sequence at a specific location, for example, at the 5' end or 3' end of the sequence optimized nucleic acid, or within a predetermined distance from those region (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 codons from the 5' end or 3' end of the sequence optimized nucleic acid).
  • a sequence optimized nucleic acid can comprise more than one subsequence having an overall codon frequency higher or lower than the overall codon frequency in the corresponding subsequence of the reference nucleic acid sequence.
  • subsequences with overall higher or lower overall codon frequencies can be organized in innumerable patterns, depending on whether the overall codon frequency is higher or lower, the length of the subsequence, the distance between subsequences, the location of the subsequences, etc.
  • Structural motifs Motifs encoded by an arrangement of nucleotides that tends to form a certain secondary structure.
  • motifs that fit into the category of disadvantageous motifs.
  • Some examples include, for example, restriction enzyme motifs, which tend to be relatively short, exact sequences such as the restriction site motifs for Xbal (TCTAGA (SEQ ID NO: 187)), EcoRI (GAATTC (SEQ ID NO: 188)), EcoRII (CCWGG (SEQ ID NO: 189), wherein W means A or T, per the IUPAC ambiguity codes), or Hindlll (AAGCTT (SEQ ID NO: 190)); enzyme sites, which tend to be longer and based on consensus not exact sequence, such in the T7 RNA polymerase (GnnnnWnCRnCTCnCnWnD (SEQ ID NO: 191), wherein n means any nucleotide, R means A or G, W means A or T, D means A or G or T but not C); structural motifs, such as GGGG repeats (Kim et al. (1991) Nature 351(6324):331-2); or other
  • nucleic acid sequence disclosed herein can be sequence optimized using methods comprising substituting at least one destabilizing motif in a reference nucleic acid sequence, and removing such disadvantageous motif or replacing it with an advantageous motif.
  • the optimization process comprises identifying advantageous and/or disadvantageous motifs in the reference nucleic sequence, wherein such motifs are, e.g., specific subsequences that can cause a loss of stability in the reference nucleic acid sequence prior or during the optimization process. For example, substitution of specific bases during optimization may generate a subsequence (motif) recognized by a restriction enzyme. Accordingly, during the optimization process the appearance of disadvantageous motifs can be monitored by comparing the sequence optimized sequence with a library of motifs known to be disadvantageous. Then, the identification of disadvantageous motifs could be used as a post-hoc filter, i.e., to determine whether a certain modification which potentially could be introduced in the reference nucleic acid sequence should be actually implemented or not.
  • motifs are, e.g., specific subsequences that can cause a loss of stability in the reference nucleic acid sequence prior or during the optimization process. For example, substitution of specific bases during optimization may generate a subsequence (motif) recognized by a restriction
  • the identification of disadvantageous motifs can be used prior to the application of the sequence optimization methods disclosed herein, i.e., the identification of motifs in the reference nucleic acid sequence and their replacement with alternative nucleic acid sequences can be used as a preprocessing step, for example, before uridine reduction.
  • the identification of disadvantageous motifs and their removal is used as an additional sequence optimization technique integrated in a multiparametric nucleic acid optimization method comprising two or more of the sequence optimization methods disclosed herein.
  • a disadvantageous motif identified during the optimization process would be removed, for example, by substituting the lowest possible number of nucleobases in order to preserve as closely as possible the original design principle(s) (e.g., low U, high frequency, etc.). See, e.g., U.S. Publ. Nos. US20140228558, US20050032730, or US20140228558, which are herein incorporated by reference in their entireties.
  • sequence optimization of a reference nucleic acid sequence can be conducted using a limited codon set, e.g., a codon set wherein less than the native number of codons is used to encode the 20 natural amino acids, a subset of the 20 natural amino acids, or an expanded set of amino acids including, for example, non-natural amino acids.
  • a limited codon set e.g., a codon set wherein less than the native number of codons is used to encode the 20 natural amino acids, a subset of the 20 natural amino acids, or an expanded set of amino acids including, for example, non-natural amino acids.
  • the genetic code is highly similar among all organisms and can be expressed in a simple table with 64 entries which would encode the 20 standard amino acids involved in protein translation plus start and stop codons.
  • the genetic code is degenerate, i.e., in general, more than one codon specifies each amino acid.
  • the amino acid leucine is specified by the UUA, UUG, CUU, CUC, CUA, or CUG codons
  • the amino acid serine is specified by UCA, UCG, UCC, UCU, AGU, or AGC codons (difference in the first, second, or third position).
  • Native genetic codes comprise 62 codons encoding naturally occurring amino acids.
  • optimized codon sets comprising less than 62 codons to encode 20 amino acids can comprise 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 codons.
  • the limited codon set comprises less than 20 codons.
  • an optimized codon set comprises as many codons as different types of amino acids are present in the protein encoded by the reference nucleic acid sequence.
  • the optimized codon set comprises 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or even 1 codon.
  • Ala can be encoded in the sequence optimized nucleic acid by 3, 2 or 1 codons; Cys can be encoded in the sequence optimized nucleic acid by 1 codon; Asp can be encoded in the sequence optimized nucleic acid by 1 codon; Glu can be encoded in the sequence optimized nucleic acid by 1 codon; Phe can be encoded in the sequence optimized nucleic acid by 1 codon; Gly can be encoded in the sequence optimized nucleic acid by 3 codons, 2 codons or 1 codon; His can be encoded in the sequence optimized nucleic acid by 1 codon; He can be encoded in the sequence optimized nucleic acid by 2 codons or 1 codon; Lys can be encoded in the sequence optimized nucleic acid by 1 codon; Leu can be encoded in the sequence optimized nucleic acid by 5 codons, 4 codons, 3 codons, 2 codons or 1 codon; Asn can be encoded in the sequence optimized nucleic acid by 1 codon; Pro
  • the sequence optimized nucleic acid is a DNA and the limited codon set consists of 20 codons, wherein each codon encodes one of 20 amino acids.
  • the sequence optimized nucleic acid is a DNA and the limited codon set comprises at least one codon selected from the group consisting of GCT, GCC, GCA, and GCG; at least a codon selected from the group consisting of CGT, CGC, CGA, CGG, AGA, and AGG; at least a codon selected from AAT or ACC; at least a codon selected from GAT or GAC; at least a codon selected from TGT or TGC; at least a codon selected from CAA or CAG; at least a codon selected from GAA or GAG; at least a codon selected from the group consisting of GGT, GGC, GGA, and GGG; at least a codon selected from CAT or CAC; at least a codon selected from the group consisting of ATT, ATC, and
  • the sequence optimized nucleic acid is an RNA (e.g., an mRNA) and the limited codon set consists of 20 codons, wherein each codon encodes one of 20 amino acids.
  • the sequence optimized nucleic acid is an RNA and the limited codon set comprises at least one codon selected from the group consisting of GCU, GCC, GCA, and GCG; at least a codon selected from the group consisting of CGU, CGC, CGA, CGG, AGA, and AGG; at least a codon selected from AAU or ACC; at least a codon selected from GAU or GAC; at least a codon selected from UGU or UGC; at least a codon selected from CAA or CAG; at least a codon selected from GAA or GAG; at least a codon selected from the group consisting of GGU, GGC, GGA, and GGG; at least a codon selected from CAU or CAC; at least a codon selected from the group consisting of GGU, GGC
  • the limited codon set has been optimized for in vivo expression of a sequence optimized nucleic acid (e.g., a synthetic mRNA) following administration to a certain tissue or cell.
  • a sequence optimized nucleic acid e.g., a synthetic mRNA
  • the optimized codon set (e.g., a 20 codon set encoding 20 amino acids) complies at least with one of the following properties:
  • the optimized codon set has a higher average G/C content than the original or native codon set;
  • the optimized codon set has a lower average U content than the original or native codon set
  • the optimized codon set is composed of codons with the highest frequency
  • the optimized codon set is composed of codons with the lowest frequency
  • At least one codon in the optimized codon set has the second highest, the third highest, the fourth highest, the fifth highest or the sixth highest frequency in the synonymous codon set. In some specific embodiments, at least one codon in the optimized codon has the second lowest, the third lowest, the fourth lowest, the fifth lowest, or the sixth lowest frequency in the synonymous codon set.
  • the term “native codon set” refers to the codon set used natively by the source organism to encode the reference nucleic acid sequence.
  • the term “original codon set” refers to the codon set used to encode the reference nucleic acid sequence before the beginning of sequence optimization, or to a codon set used to encode an optimized variant of the reference nucleic acid sequence at the beginning of a new optimization iteration when sequence optimization is applied iteratively or recursively.
  • 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons in the codon set are those with the highest frequency.
  • 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons in the codon set are those with the lowest frequency.
  • 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons in the codon set are those with the highest uridine content. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons in the codon set are those with the lowest uridine content.
  • the average G/C content (absolute or relative) of the codon set is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% higher than the average G/C content (absolute or relative) of the original codon set.
  • the average G/C content (absolute or relative) of the codon set is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% lower than the average G/C content (absolute or relative) of the original codon set.
  • the uracil content (absolute or relative) of the codon set is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% higher than the average uracil content (absolute or relative) of the original codon set.
  • the uracil content (absolute or relative) of the codon set is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% lower than the average uracil content (absolute or relative) of the original codon set.
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • a sequence optimized nucleic acid disclosed herein can be can be tested to determine whether at least one nucleic acid sequence property (e.g., stability when exposed to nucleases) or expression property has been improved with respect to the non-sequence optimized nucleic acid.
  • expression property refers to a property of a nucleic acid sequence either in vivo (e.g., translation efficacy of a synthetic mRNA after administration to a subject in need thereof) or in vitro (e.g., translation efficacy of a synthetic mRNA tested in an in vitro model system). Expression properties include but are not limited to the amount of protein produced by an mRNA after administration, and the amount of soluble or otherwise functional protein produced.
  • sequence optimized nucleic acids disclosed herein can be evaluated according to the viability of the cells expressing a protein encoded by a sequence optimized nucleic acid sequence (e.g., a RNA, e.g., an mRNA) disclosed herein.
  • a plurality of sequence optimized nucleic acids disclosed herein e.g., a RNA, e.g., an mRNA
  • a property of interest for example an expression property in an in vitro model system, or in vivo in a target tissue or cell.
  • the desired property of the polynucleotide is an intrinsic property of the nucleic acid sequence.
  • the nucleotide sequence e.g., a RNA, e.g., an mRNA
  • the nucleotide sequence can be sequence optimized for in vivo or in vitro stability.
  • the nucleotide sequence can be sequence optimized for expression in a particular target tissue or cell.
  • the nucleic acid sequence is sequence optimized to increase its plasma half by preventing its degradation by endo and exonucleases.
  • the nucleic acid sequence is sequence optimized to increase its resistance to hydrolysis in solution, for example, to lengthen the time that the sequence optimized nucleic acid or a pharmaceutical composition comprising the sequence optimized nucleic acid can be stored under aqueous conditions with minimal degradation.
  • sequence optimized nucleic acid can be optimized to increase its resistance to hydrolysis in dry storage conditions, for example, to lengthen the time that the sequence optimized nucleic acid can be stored after lyophilization with minimal degradation.
  • the desired property of the polynucleotide is the level of expression of an IL-12A polypeptide, an IL-12B polypeptide, or both IL-12A and IL-12B polypeptides and a membrane domain encoded by a sequence optimized sequence disclosed herein.
  • Protein expression levels can be measured using one or more expression systems.
  • expression can be measured in cell culture systems, e.g., CHO cells or HEK293 cells.
  • expression can be measured using in vitro expression systems prepared from extracts of living cells, e.g., rabbit reticulocyte lysates, or in vitro expression systems prepared by assembly of purified individual components.
  • the protein expression is measured in an in vivo system, e.g., mouse, rabbit, monkey, etc.
  • protein expression in solution form can be desirable.
  • a reference sequence can be sequence optimized to yield a sequence optimized nucleic acid sequence having optimized levels of expressed proteins in soluble form.
  • Levels of protein expression and other properties such as solubility, levels of aggregation, and the presence of truncation products (i.e., fragments due to proteolysis, hydrolysis, or defective translation) can be measured according to methods known in the art, for example, using electrophoresis (e.g., native or SDS-PAGE) or chromatographic methods (e.g., HPLC, size exclusion chromatography, etc.).
  • the expression of heterologous therapeutic proteins encoded by a nucleic acid sequence can have deleterious effects in the target tissue or cell, reducing protein yield, or reducing the quality of the expressed product (e.g., due to the presence of protein fragments or precipitation of the expressed protein in inclusion bodies), or causing toxicity.
  • the sequence optimization of a nucleic acid sequence disclosed herein e.g., a nucleic acid sequence encoding an IL- 12 polypeptide and/or a nucleic acid sequence encoding a membrane domain, can be used to increase the viability of target cells expressing the protein encoded by the sequence optimized nucleic acid.
  • Heterologous protein expression can also be deleterious to cells transfected with a nucleic acid sequence for autologous or heterologous transplantation. Accordingly, in some embodiments of the present disclosure the sequence optimization of a nucleic acid sequence disclosed herein can be used to increase the viability of target cells expressing the protein encoded by the sequence optimized nucleic acid sequence. Changes in cell or tissue viability, toxicity, and other physiological reaction can be measured according to methods known in the art. d. Reduction of Immune and/or Inflammatory Response
  • the administration of a sequence optimized nucleic acid encoding a tethered IL- 12 polypeptide as disclosed herein, including functional fragments and variants thereof, may trigger an immune response, which could be caused by (i) the therapeutic agent (e.g., an mRNA comprising a nucleic acid sequence encoding a tethered IL- 12 polypeptide), or (ii) the expression product of such therapeutic agent (e.g., the tethered IL- 12 polypeptide encoded by the mRNA), or (iv) a combination thereof.
  • the therapeutic agent e.g., an mRNA comprising a nucleic acid sequence encoding a tethered IL- 12 polypeptide
  • the expression product of such therapeutic agent e.g., the tethered IL- 12 polypeptide encoded by the mRNA
  • nucleic acid sequence e.g., an mRNA
  • sequence optimization of nucleic acid sequence can be used to decrease an immune or inflammatory response triggered by the administration of a nucleic acid encoding a tethered IL- 12 polypeptide or by the expression product of the tethered IL-12 polypeptide encoded by such nucleic acid.
  • an inflammatory response can be measured by detecting increased levels of one or more inflammatory cytokines using methods known in the art, e.g., ELISA.
  • inflammatory cytokine refers to cytokines that are elevated in an inflammatory response.
  • inflammatory cytokines include interleukin-6 (IL-6), CXCL1 (chemokine (C-X-C motif) ligand 1 ; also known as GROoc, interferon- ⁇ (IFNy), tumor necrosis factor a (TNF ), interferon ⁇ -induced protein 10 (IP- 10), or granulocyte-colony stimulating factor (G-CSF).
  • IFNy interleukin-6
  • CXCL1 chemokine (C-X-C motif) ligand 1
  • GROoc interferon- ⁇
  • TNF tumor necrosis factor a
  • IP- 10 interferon ⁇ -induced protein 10
  • G-CSF granulocyte-colony stimulating factor
  • inflammatory cytokines includes also other cytokines associated with inflammatory responses known in the art, e.g., interleukin- 1 (IL- 1), interleukin-8 (IL-8), interleukin- 13 (11- 13), interferon a (IFN-a), etc. 12.
  • IL- 1 interleukin- 1
  • IL-8 interleukin-8
  • IFN-a interferon a
  • the disclosure includes modified polynucleotides comprising a polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding an IL- 12 polypeptide and a nucleotide sequence encoding a membrane domain).
  • the modified polynucleotides can be chemically modified and/or structurally modified.
  • the polynucleotides of the present disclosure are chemically and/or structurally modified the polynucleotides can be referred to as "modified polynucleotides.”
  • nucleosides and nucleotides of a polynucleotide e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • a “nucleoside” refers to a compound containing a sugar molecule (e.g. , a pentose or ribose) or a derivative thereof in combination with an organic base (e.g. , a purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleobase”).
  • a “nucleotide” refers to a nucleoside including a phosphate group.
  • Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non- natural nucleosides.
  • Polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.
  • modified polynucleotides disclosed herein can comprise various distinct modifications.
  • the modified polynucleotides contain one, two, or more (optionally different) nucleoside or nucleotide modifications.
  • a modified polynucleotide, introduced to a cell may exhibit one or more desirable properties, e.g., improved protein expression, reduced immunogenicity, or reduced degradation in the cell, as compared to an unmodified polynucleotide.
  • a polynucleotide of the present disclosure e.g., a polynucleotide comprising a nucleotide sequence encoding an IL- 12 polypeptide and a nucleotide sequence encoding a membrane domain
  • a "structural" modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or
  • the polynucleotides of the present disclosure ⁇ e.g., a polynucleotide comprising a nucleotide sequence encoding an IL-12 polypeptide and a nucleotide sequence encoding a membrane domain
  • the terms "chemical modification” or, as appropriate, “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or deoxyribonucleosides in one or more of their position, pattern, percent or population, including, but not limited to, its nucleobase, sugar, backbone, or any combination thereof.
  • these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5 '-terminal mRNA cap moieties.
  • the polynucleotides of the disclosure can have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications produced by downward titration of the same starting modification in all or any of the same nucleoside type, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation, such as where all uridines are replaced by a uridine analog, e.g., 5-methoxyuridine.
  • the polynucleotides can have a uniform chemical modification of two, three, or four of the same nucleoside type throughout the entire polynucleotide (such as all uridines and/or all cytidines, etc. are modified in the same way).
  • Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a nonstandard base and a standard base or between two complementary non-standard base structures.
  • non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base/sugar or linker may be incorporated into polynucleotides of the present disclosure.
  • RNA polynucleotides such as mRNA polynucleotides
  • Modifications of polynucleotides include, but are not limited to the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6- methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; l,2'-0- dimethyladenosine; 1-methyladenosine; 2'-0-methyladenosine; 2'-0-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine;
  • N6-(cis-hydroxyisopentenyl)adenosine N6,2'-0-dimethyladenosine; N6,2'- O-dimethyladenosine; N6,N6,2'-0-trimethyladenosine; N6,N6-dimethyladenosine; N6- acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine; N6-methyl-N6- threonylcarbamoyladenosine; 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza- adenosine; Nl-methyl-adenosine; N6, N6 (dimethyl) adenine; N6-cis-hydroxy-isopentenyl- adenosine; a-thio-adenosine; 2 (amino)adenine; 2 (amino)adenine; 2 (amin
  • Carbamoylmethyluridine TP 5-methoxycarbonylmethyl-2'-0-methyluridine; 5- methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methyluridine,), 5- methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5- methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-Methyldihydrouridine; 5- Oxyacetic acid- Uridine TP; 5-Oxyacetic acid-methyl ester- Uridine TP; Nl-methyl-pseudo-uridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-Amino-3-carboxypropyl)- Uridine TP; 5-(iso-Pentenylaminomethyl)- 2-thiouridine TP; 5-(iso-Pentenylaminomethyl)-2'-0- methyluridine TP
  • the polynucleotide e.g. , RNA polynucleotide, such as mRNA polynucleotide
  • the polynucleotide includes a combination of at least two (e.g. , 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • the mRNA comprises at least one chemically modified nucleoside.
  • the at least one chemically modified nucleoside is selected from the group consisting of pseudouridine ( ⁇ ), Nl-methylpseudo uridine ( ⁇ ), 2-thiouridine (s2U), 4'-thiouridine, 5-methylcytosine, 2-thio- 1 -methyl- 1-deaza-pseudouridine, 2-thio- 1-methyl-pseudouridine, 2-thio-5- aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy- 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio- 1-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2'-
  • the at least one chemically modified nucleoside is selected from the group consisting of pseudouridine, Nl-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and a combination thereof.
  • the polynucleotide e.g. , RNA polynucleotide, such as mRNA polynucleotide
  • the polynucleotide includes a combination of at least two (e.g. , 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • the chemical modification is at nucleobases in the polynucleotides (e.g. , RNA polynucleotide, such as mRNA polynucleotide).
  • a polynucleotide as disclosed herein comprises at least one chemically modified nucleobase.
  • the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil ( ⁇ ), Nl-methylpseudouracil 2-thiouracil (s2U), 4'-
  • the nucleobases in a polynucleotide as disclosed herein are chemically modified by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 95%, at least 99%, or 100%.
  • the chemically modified nucleobases are selected from the group consisting of uracil, adenine, cytosine, guanine, and any combination thereof.
  • the uracils in a polynucleotide disclosed herein are chemically modified by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 95%, at least 99%, or 100%.
  • the adenines in a polynucleotide disclosed herein are chemically modified by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 95%, at least 99%, or 100%.
  • the cytosines in a polynucleotide disclosed herein are chemically modified by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 95%, at least 99%, or 100%.
  • the guanines in a polynucleotide disclosed herein are chemically modified by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 95%, at least 99%, or 100%.
  • the polynucleotide e.g. , RNA polynucleotide, such as mRNA polynucleotide
  • the polynucleotide includes a combination of at least two (e.g. , 2, 3, 4 or more) of modified nucleobases.
  • modified nucleobases in the polynucleotide e.g.
  • RNA polynucleotide such as mRNA polynucleotide
  • RNA polynucleotide are selected from the group consisting of 1-methyl- pseudouridine ( ⁇ ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine ( ⁇ ), ⁇ - thio-guanosine and a-thio-adenosine.
  • the polynucleotide includes a combination of at least two (e.g. , 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • the polynucleotide (e.g. , RNA polynucleotide, such as mRNA polynucleotide) comprises pseudouridine ( ⁇ ) and 5-methyl-cytidine (m5C).
  • the polynucleotide (e.g. , RNA polynucleotide, such as mRNA polynucleotide) comprises 1-methyl- pseudouridine ( ⁇ ).
  • the polynucleotide e.g. , RNA polynucleotide, such as mRNA polynucleotide
  • the polynucleotide (e.g. , RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine (s2U). In some embodiments, the polynucleotide (e.g. , RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g. , RNA polynucleotide, such as mRNA polynucleotide) comprises methoxy-uridine (mo5U). In some embodiments, the polynucleotide (e.g.
  • RNA polynucleotide such as mRNA polynucleotide
  • RNA polynucleotide comprises 5-methoxy-uridine (mo5U) and 5-methyl- cytidine (m5C).
  • the polynucleotide e.g. , RNA polynucleotide, such as mRNA polynucleotide
  • the polynucleotide comprises 2'-0-methyl uridine.
  • the polynucleotide e.g. , RNA polynucleotide, such as mRNA polynucleotide
  • the polynucleotide e.g.
  • RNA polynucleotide such as mRNA polynucleotide
  • N6-methyl-adenosine m6A
  • the polynucleotide e.g. , RNA polynucleotide, such as mRNA polynucleotide
  • N6-methyl- adenosine m6A
  • 5-methyl-cytidine m5C
  • the polynucleotide e.g. , RNA polynucleotide, such as mRNA polynucleotide
  • RNA polynucleotide is uniformly modified (e.g. , fully modified, modified throughout the entire sequence) for a particular modification.
  • a polynucleotide can be uniformly modified with 5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C).
  • m5C 5-methyl-cytidine
  • a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as any of those set forth above.
  • the modified nucleobase is a modified cytosine.
  • nucleobases and nucleosides having a modified cytosine include N4-acetyl-cytidine (ac4C), 5- methyl-cytidine (m5C), 5-halo-cytidine (e.g. , 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.
  • a modified nucleobase is a modified uridine.
  • Example nucleobases and nucleosides having a modified uridine include 5-cyano uridine or 4'-thio uridine.
  • a modified nucleobase is a modified adenine.
  • Example nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl-adenosine (mlA), 2- methyl- adenine (m2A), N6-methyl-adenine (m6A), and 2,6-Diaminopurine.
  • a modified nucleobase is a modified guanine.
  • Example nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (mil), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQO), 7- aminomethyl-7-deaza-guanosine (preQl), 7-methyl-guanosine (m7G), 1-methyl-guanosine (mlG), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.
  • the nucleobases, sugar, backbone, or any combination thereof in a polynucleotide, nucleic acid sequence, and/or ORF as disclosed herein are chemically modified by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.
  • the polynucleotides can include any useful linker between the nucleosides, including subunit linkers, membrane domain linkers, and heterologous polypeptide linkers as disclosed elsewhere herein.
  • linkers, including backbone modifications, that are useful in the composition of the present disclosure include, but are not limited to the following: 3'- alkylene phosphonates, 3'-amino phosphoramidate, alkene containing backbones, aminoalkylphosphoramidates, aminoalkylphosphotriesters, boranophosphates, -CH2-0-N(CH3)-CH2- , -CH2-N(CH3)-N(CH3)-CH2-, -CH2-NH-CH2-, chiral phosphonates, chiral phosphorothioates, formacetyl and thioformacetyl backbones, methylene (methylimino), methylene formacetyl and thioformacetyl backbones, methyleneimino and methylenehydr
  • modified nucleosides and nucleotides which can be incorporated into a polynucleotide (e.g., RNA or mRNA, as described herein), can be modified on the sugar of the ribonucleic acid.
  • a polynucleotide e.g., RNA or mRNA, as described herein
  • the 2' hydroxyl group can be modified or replaced with a number of different substituents.
  • substitutions at the 2'-position include, but are not limited to, H, halo, optionally substituted C 1-6 alkyl; optionally substituted C 1-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C 6 -io aryloxy; optionally substituted C6-10 aryl-Ci-6 alkoxy, optionally substituted Ci-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), -0(CH 2 CH20)nCH2CH 2 OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from
  • RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen.
  • modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4- membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7- membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar.
  • Such sugar modifications are taught International Patent Publication Nos. WO2013052523 and WO2014093924, the contents of each of which are incorporated herein by reference in their entireties.
  • polynucleotides of the disclosure can include a combination of modifications to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more modifications described herein.
  • modified nucleotides can be used to form the polynucleotides of the disclosure.
  • the modified nucleotides can be completely substituted for the natural nucleotides of the polynucleotides of the disclosure.
  • the natural nucleotide uridine can be substituted with a modified nucleoside described herein.
  • the natural nucleotide uridine can be partially substituted or replaced (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%) with at least one of the modified nucleoside disclosed herein.
  • Any combination of base/sugar or linker can be incorporated into the polynucleotides of the disclosure and such modifications are taught in International Patent Publications WO2013052523 and WO2014093924, and U.S. Publ. Nos. US 20130115272 and US20150307542, the contents of each of which are incorporated herein by reference in its entirety.
  • Untranslated regions are nucleic acid sections of a polynucleotide before a start codon (5'UTR) and after a stop codon (3'UTR) that are not translated.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • UTR e.g., a 5'UTR or functional fragment thereof, a 3 'UTR or functional fragment thereof, or a combination thereof.
  • a UTR can be homologous or heterologous to the coding region in a polynucleotide.
  • the UTR is homologous to the nucleic acid sequence encoding the IL- 12 polypeptide. In some embodiments, the UTR is heterologous to the nucleic acide sequence encoding the IL-12 polypeptide.
  • the polynucleotide comprises two or more 5'UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences. In some embodiments, the polynucleotide comprises two or more 3 'UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences.
  • the 5 'UTR or functional fragment thereof, 3 ' UTR or functional fragment thereof, or any combination thereof is sequence optimized.
  • the 5 'UTR or functional fragment thereof, 3 ' UTR or functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, e.g., 1 methylpseudouridine or 5-methoxyuracil.
  • UTRs can have features that provide a regulatory role, e.g., increased or decreased stability, localization and/or translation efficiency.
  • a polynucleotide comprising a UTR can be administered to a cell, tissue, or organism, and one or more regulatory features can be measured using routine methods.
  • a functional fragment of a 5'UTR or 3'UTR comprises one or more regulatory features of a full length 5' or 3' UTR, respectively.
  • Natural 5'UTRs bear features that play roles in translation initiation. They harbor signatures like Kozak sequences that are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG (SEQ ID NO: 216), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'. 5'UTRs also have been known to form secondary structures that are involved in elongation factor binding.
  • liver-expressed mRNA such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, can enhance expression of polynucleotides in hepatic cell lines or liver.
  • 5'UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g., Tie- 1, CD36), for myeloid cells (e.g., C/EBP, AML1, G-CSF, GM-CSF, CD l ib, MSR, Fr- 1, i-NOS), for leukocytes (e.g., CD45, CD 18), for adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (e.g., SP-A/B/C/D).
  • muscle e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin
  • endothelial cells e.g., Tie- 1, CD36
  • myeloid cells e.g.,
  • UTRs are selected from a family of transcripts whose proteins share a common function, structure, feature or property.
  • an encoded polypeptide can belong to a family of proteins (i.e., that share at least one function, structure, feature, localization, origin, or expression pattern), which are expressed in a particular cell, tissue or at some time during development.
  • the UTRs from any of the genes or mRNA can be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.
  • the 5'UTR and the 3'UTR can be heterologous. In some embodiments, the 5'UTR can be derived from a different species than the 3'UTR. In some embodiments, the 3'UTR can be derived from a different species than the 5'UTR.
  • Exemplary UTRs of the application include, but are not limited to, one or more 5'UTR and/or 3 'UTR derived from the nucleic acid sequence of: a globin, such as an a- or ⁇ -globin (e.g., a Xenopus, mouse, rabbit, or human globin); a strong Kozak translational initiation signal; a CYBA (e.g., human cytochrome b-245 a polypeptide); an albumin (e.g., human albumin7); a HSD17B4 (hydroxysteroid (17- ⁇ ) dehydrogenase); a virus (e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitis virus (VEEV), a Dengue virus, a cytomegalovirus (CMV) (e.g., CMV immediate early 1 (IE1)), a hepatitis virus (e.g., hepatitis B virus), a Sindbis virus
  • the 5'UTR is selected from the group consisting of a ⁇ -globin 5 'UTR; a 5'UTR containing a strong Kozak translational initiation signal; a cytochrome b-245 a polypeptide (CYBA) 5'UTR; a hydroxysteroid (17- ⁇ ) dehydrogenase (HSD17B4) 5'UTR; a Tobacco etch virus (TEV) 5'UTR; a Vietnamese etch virus (TEV) 5'UTR; a decielen equine encephalitis virus (TEEV) 5'UTR; a 5' proximal open reading frame of rubella virus (RV) RNA encoding nonstructural proteins; a Dengue virus (DEN) 5'UTR; a heat shock protein 70 (Hsp70) 5'UTR; a eIF4G 5'UTR; a GLUT1 5'UTR; functional fragments thereof and any combination thereof.
  • CYBA cytochrome b
  • the 3'UTR is selected from the group consisting of a ⁇ -globin 3'UTR; a CYBA 3'UTR; an albumin 3'UTR; a growth hormone (GH) 3'UTR; a VEEV 3'UTR; a hepatitis B virus (HBV) 3'UTR; a-globin 3 'UTR; a DEN 3'UTR; a PAV barley yellow dwarf virus (BYDV- PAV) 3'UTR; an elongation factor 1 al (EEF1A1) 3'UTR; a manganese superoxide dismutase (MnSOD) 3'UTR; a ⁇ subunit of mitochondrial H(+)-ATP synthase ( ⁇ -mRNA) 3'UTR; a GLUT1 3'UTR; a MEF2A 3'UTR; a ⁇ -Fl-ATPase 3'UTR; functional fragments thereof and combinations thereof.
  • Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the disclosure.
  • a UTR can be altered relative to a wild type or native UTR to produce a variant UTR, e.g., by changing the orientation or location of the UTR relative to the ORF; or by inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.
  • variants of 5' or 3 ' UTRs can be utilized, for example, mutants of wild type UTRs, or variants wherein one or more nucleotides are added to or removed from a terminus of the UTR.
  • one or more synthetic UTRs can be used in combination with one or more non- synthetic UTRs. See, e.g., Mandal and Rossi, Nat. Protoc. 2013 8(3):568-82, the contents of which are incorporated herein by reference in their entirety, and sequences available at www.addgene.org/Derrick_Rossi/, last accessed April 16, 2016. UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected or can be altered in orientation or location. Hence, a 5' and/or 3 ' UTR can be inverted, shortened, lengthened, or combined with one or more other 5' UTRs or 3 ' UTRs.
  • the polynucleotide comprises multiple UTRs, e.g., a double, a triple or a quadruple 5'UTR or 3'UTR.
  • a double UTR comprises two copies of the same UTR either in series or substantially in series.
  • a double beta-globin 3 'UTR can be used (see US2010/0129877, the contents of which are incorporated herein by reference in its entirety).
  • the polynucleotides of the disclosure comprise a 5'UTR and/or a 3'UTR selected from any of the UTRs disclosed herein.
  • the 5'UTR comprises a sequence selected from the group consisting of: SEQ ID NOs: 55-63 and 82-97.
  • the 3'UTR comprises a sequence selected from the group consisting of: SEQ ID NOs: 64-81.
  • the 5'UTR and/or 3'UTR sequence of the disclosure comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of 5'UTR sequences comprising any of the 5'UTR sequences disclosed herein and/or 3'UTR sequences comprises any of the 3'UTR sequences disclosed herein, and any combination thereof.
  • the 3' UTR sequence comprises one or more miRNA binding sites, e.g. , miR-122 binding sites, or any other heterologous nucleotide sequences therein, without disrupting the function of the 3' UTR.
  • Some examples of 3' UTR sequences comprising a miRNA binding site are set forth in SEQ ID NOs: 222-224.
  • the 3' UTR sequence comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 222-224.
  • the 3' UTR sequence comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 222.
  • the 3' UTR sequence comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 223.
  • the 3' UTR sequence comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 224.
  • the polynucleotides of the disclosure can comprise combinations of features.
  • the ORF can be flanked by a 5 'UTR that comprises a strong Kozak translational initiation signal and/or a 3 'UTR comprising an oligo(dT) sequence for templated addition of a poly- A tail.
  • a 5 'UTR can comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different UTRs (see, e.g., US2010/0293625, herein incorporated by reference in its entirety).
  • non-UTR sequences can be used as regions or subregions within the polynucleotides of the disclosure.
  • introns or portions of intron sequences can be incorporated into the polynucleotides of the disclosure. Incorporation of intronic sequences can increase protein production as well as polynucleotide expression levels.
  • the polynucleotide of the disclosure comprises an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e.g., Yakubov et ah, Biochem. Biophys. Res. Commun. 2010 394(1): 189-193, the contents of which are incorporated herein by reference in their entirety).
  • ITR internal ribosome entry site
  • the polynucleotide comprises an IRES instead of a 5'UTR sequence. In some embodiments, the polynucleotide comprises an ORF and a viral capsid sequence. In some embodiments, the polynucleotide comprises a synthetic 5'UTR in combination with a non-synthetic 3'UTR.
  • the UTR can also include at least one translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements (collectively, "TEE," which refers to nucleic acid sequences that increase the amount of polypeptide or protein produced from a polynucleotide.
  • TEE translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements
  • the TEE can include those described in US2009/0226470, incorporated herein by reference in its entirety, and others known in the art.
  • the TEE can be located between the transcription promoter and the start codon.
  • the 5'UTR comprises a TEE.
  • a TEE is a conserved element in a UTR that can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or cap-independent translation.
  • the TEE comprises the TEE sequence in the 5 '-leader of the Gtx homeodomain protein. See Chappell et al., PNAS 2004 101:9590-9594, incorporated herein by reference in its entirety.
  • the polynucleotide of the invention comprises one or multiple copies of a TEE.
  • the TEE in a translational enhancer polynucleotide can be organized in one or more sequence segments.
  • a sequence segment can harbor one or more of the TEEs provided herein, with each TEE being present in one or more copies.
  • multiple sequence segments are present in a translational enhancer polynucleotide, they can be homogenous or heterogeneous.
  • the multiple sequence segments in a translational enhancer polynucleotide can harbor identical or different types of the TEE provided herein, identical or different number of copies of each of the TEE, and/or identical or different organization of the TEE within each sequence segment.
  • the polynucleotide of the invention comprises a translational enhancer polynucleotide sequence.
  • TEE sequences are described in U.S. Publication 2014/0200261, the contents of which are incorporated herein by reference in their entirety.
  • the disclosure provides polynucleotides comprising a modification (e.g., an RNA element), wherein the modification provides a desired translational regulatory activity.
  • a modification e.g., an RNA element
  • the disclosure provides a polynucleotide comprising a 5' untranslated region (UTR), an initiation codon, a full open reading frame encoding a polypeptide, a 3' UTR, and at least one modification, wherein the at least one modification provides a desired translational regulatory activity, for example, a modification that promotes and/or enhances the translational fidelity of mRNA translation.
  • the desired translational regulatory activity is a cis-acting regulatory activity.
  • the desired translational regulatory activity is an increase in the residence time of the 43S pre-initiation complex (PIC) or ribosome at, or proximal to, the initiation codon. In some embodiments, the desired translational regulatory activity is an increase in the initiation of polypeptide synthesis at or from the initiation codon. In some embodiments, the desired translational regulatory activity is an increase in the amount of polypeptide translated from the full open reading frame. In some embodiments, the desired translational regulatory activity is an increase in the fidelity of initiation codon decoding by the PIC or ribosome. In some embodiments, the desired translational regulatory activity is inhibition or reduction of leaky scanning by the PIC or ribosome.
  • the desired translational regulatory activity is a decrease in the rate of decoding the initiation codon by the PIC or ribosome. In some embodiments, the desired translational regulatory activity is inhibition or reduction in the initiation of polypeptide synthesis at any codon within the mRNA other than the initiation codon. In some embodiments, the desired translational regulatory activity is inhibition or reduction of the amount of polypeptide translated from any open reading frame within the mRNA other than the full open reading frame. In some embodiments, the desired translational regulatory activity is inhibition or reduction in the production of aberrant translation products. In some embodiments, the desired translational regulatory activity is a combination of one or more of the foregoing translational regulatory activities.
  • the present disclosure provides a polynucleotide, e.g., an mRNA, comprising an RNA element that comprises a sequence and/or an RNA secondary structure(s) that provides a desired translational regulatory activity as described herein.
  • the mRNA comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that promotes and/or enhances the translational fidelity of mRNA translation.
  • the mRNA comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that provides a desired translational regulatory activity, such as inhibiting and/or reducing leaky scanning.
  • the disclosure provides an mRNA that comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that inhibits and/or reduces leaky scanning thereby promoting the translational fidelity of the mRNA.
  • the RNA element comprises natural and/or modified nucleotides. In some embodiments, the RNA element comprises of a sequence of linked nucleotides, or derivatives or analogs thereof, that provides a desired translational regulatory activity as described herein. In some embodiments, the RNA element comprises a sequence of linked nucleotides, or derivatives or analogs thereof, that forms or folds into a stable RNA secondary structure, wherein the RNA secondary structure provides a desired translational regulatory activity as described herein.
  • RNA elements can be identified and/or characterized based on the primary sequence of the element (e.g., GC-rich element), by RNA secondary structure formed by the element (e.g.
  • RNA molecules e.g., located within the 5' UTR of an mRNA
  • RNA molecule e.g., located within the 5' UTR of an mRNA
  • biological function and/or activity of the element e.g., "translational enhancer element”
  • the disclosure provides an mRNA having one or more structural modifications that inhibits leaky scanning and/or promotes the translational fidelity of mRNA translation, wherein at least one of the structural modifications is a GC-rich RNA element.
  • the disclosure provides an mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5' UTR of the mRNA.
  • the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5' UTR of the mRNA. In another embodiment, the GC-rich RNA element is located 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5' UTR of the mRNA.
  • the disclosure provides a GC-rich RNA element which comprises a sequence of 3-30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60-70% cytosine, 50%-60% cytosine, 40-50% cytosine, 30-40% cytosine bases.
  • the disclosure provides a GC-rich RNA element which comprises a sequence of 3-30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.
  • the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60- 70% cytosine, 50%-60% cytosine, 40-50% cytosine, or 30-40% cytosine.
  • the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.
  • the disclosure provides an mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5' UTR of the mRNA, wherein the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5' UTR of the mRNA, and wherein the GC-rich RNA element comprises a sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is >50% cytosine.
  • at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5
  • the sequence composition is >55% cytosine, >60% cytosine, >65% cytosine, >70% cytosine, >75% cytosine, >80% cytosine, >85% cytosine, or >90% cytosine.
  • the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5' UTR of the mRNA. In another embodiment, the GC-rich RNA element is located about 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5' UTR of the mRNA.
  • the disclosure provides an mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence set forth in SEQ ID NO: 258, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence as set forth in SEQ ID NO: 258 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence as set forth in SEQ ID NO: 258 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence as set forth in SEQ ID NO: 258 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the disclosure provides an mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence as set forth SEQ ID NO: 259, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence as set forth SEQ ID NO: 259 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence as set forth SEQ ID NO: 259 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence as set forth SEQ ID NO: 259 located 1-3, 3-5, 5-7, 7-9, 9- 12, or 12-15 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the disclosure provides an mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence as set forth in SEQ ID NO: 260, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence as set forth in SEQ ID NO: 260 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence as set forth in SEQ ID NO: 260 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the GC-rich element comprises the sequence as set forth in SEQ ID NO: 260 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA.
  • the disclosure provides an mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence set forth in SEQ ID NO: 258, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5' UTR of the mRNA, wherein the 5' UTR comprises the sequence set forth in SEQ ID NO: 261.
  • the GC-rich element comprises the sequence set forth in SEQ ID NO: 258 located immediately adjacent to and upstream of the Kozak consensus sequence in a 5' UTR sequence described herein. In some embodiments, the GC-rich element comprises the sequence set forth in SEQ ID NO: 258 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA, wherein the 5' UTR comprises the sequence shown in SEQ ID NO: 261.
  • the GC-rich element comprises the sequence set forth in SEQ ID NO: 258 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5' UTR of the mRNA, wherein the 5' UTR comprises the sequence set forth in SEQ ID NO: 261.
  • the 5' UTR comprises the sequence set forth in SEQ ID NO: 262.
  • the 5' UTR comprises the sequence set forth in SEQ ID NO: 263.
  • the disclosure provides an mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a stable RNA secondary structure comprising a sequence of nucleotides, or derivatives or analogs thereof, linked in an order which forms a hairpin or a stem- loop.
  • the stable RNA secondary structure is upstream of the Kozak consensus sequence.
  • the stable RNA secondary structure is located about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides upstream of the Kozak consensus sequence.
  • the stable RNA secondary structure is located about 20, about 15, about 10 or about 5 nucleotides upstream of the Kozak consensus sequence.
  • the stable RNA secondary structure is located about 5, about 4, about 3, about 2, about 1 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 15-30, about 15-20, about 15-25, about 10-15, or about 5-10 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located 12-15 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure has a deltaG of about -30 kcal/mol, about -20 to -30 kcal/mol, about -20 kcal/mol, about -10 to -20 kcal/mol, about -10 kcal/mol, about -5 to -10 kcal/mol.
  • the modification is operably linked to an open reading frame encoding a polypeptide and wherein the modification and the open reading frame are heterologous.
  • the sequence of the GC-rich RNA element is comprised exclusively of guanine (G) and cytosine (C) nucleobases.
  • RNA elements that provide a desired translational regulatory activity as described herein can be identified and characterized using known techniques, such as ribosome profiling.
  • Ribosome profiling is a technique that allows the determination of the positions of PICs and/or ribosomes bound to mRNAs (see e.g., Ingolia et al., (2009) Science 324(5924):218-23, incorporated herein by reference). The technique is based on protecting a region or segment of mRNA, by the PIC and/or ribosome, from nuclease digestion. Protection results in the generation of a 30-bp fragment of RNA termed a 'footprint' .
  • RNA footprints can be analyzed by methods known in the art (e.g., RNA-seq).
  • the footprint is roughly centered on the A-site of the ribosome. If the PIC or ribosome dwells at a particular position or location along an mRNA, footprints generated at these position would be relatively common. Studies have shown that more footprints are generated at positions where the PIC and/or ribosome exhibits decreased processivity and fewer footprints where the PIC and/or ribosome exhibits increased processivity (Gardin et al., (2014) eLife
  • residence time or the time of occupancy of a the PIC or ribosome at a discrete position or location along an polynucleotide comprising any one or more of the RNA elements described herein is determined by ribosome profiling.
  • Sensor sequences include, for example, microRNA (miRNA) binding sites, transcription factor binding sites, structured mRNA sequences and/or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof.
  • miRNA microRNA
  • Non-limiting examples of sensor sequences are described in U.S. Publication 2014/0200261, the contents of which are incorporated herein by reference in their entirety.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • the sensor sequence is a miRNA binding site.
  • a miRNA is a 19-25 nucleotide long noncoding RNA that binds to a polynucleotide and down-regulates gene expression either by reducing stability or by inhibiting translation of the polynucleotide.
  • a miRNA sequence comprises a "seed" region, i.e., a sequence in the region of positions 2-8 of the mature miRNA.
  • a miRNA seed can comprise positions 2-8 or 2-7 of the mature miRNA.
  • a miRNA seed can comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature miRNA), wherein the seed-complementary site in the corresponding miRNA binding site is flanked by an adenosine (A) opposed to miRNA position 1.
  • A adenosine
  • a miRNA seed can comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature miRNA), wherein the seed- complementary site in the corresponding miRNA binding site is flanked by an adenosine (A) opposed to miRNA position 1.
  • A adenosine
  • miRNA profiling of the target cells or tissues can be conducted to determine the presence or absence of miRNA in the cells or tissues.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • microRNA binding site refers to a sequence within a polynucleotide, e.g., within a DNA or within an RNA transcript, including in the 5'UTR and/or 3 'UTR, that has sufficient complementarity to all or a region of a miRNA to interact with, associate with or bind to the miRNA.
  • a polynucleotide of the disclosure further comprises a miRNA binding site.
  • a 5'UTR and/or 3'UTR of the polynucleotide comprises a miRNA binding site.
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • a miRNA binding site having sufficient complementarity to a miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated regulation of a polynucleotide, e.g., miRNA-mediated translational repression or degradation of the polynucleotide.
  • a miRNA binding site having sufficient complementarity to the miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated degradation of the polynucleotide, e.g., miRNA-guided RNA-induced silencing complex (RlSC)-mediated cleavage of mRNA.
  • RlSC miRNA-guided RNA-induced silencing complex
  • the miRNA binding site can have complementarity to, for example, a 19-25 nucleotide miRNA sequence, to a 19-23 nucleotide miRNA sequence, or to a 22 nucleotide miRNA sequence.
  • a miRNA binding site can be complementary to only a portion of a miRNA, e.g., to a portion less than 1, 2, 3, or 4 nucleotides of the full length of a naturally-occurring miRNA sequence.
  • Full or complete complementarity e.g., full complementarity or complete complementarity over all or a significant portion of the length of a naturally-occurring miRNA is preferred when the desired regulation is mRNA degradation.
  • a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with a miRNA seed sequence. In some embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA seed sequence. In some embodiments, a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with a miRNA sequence. In some embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA sequence. In some embodiments, a miRNA binding site has complete complementarity with a miRNA sequence but for 1, 2, or 3 nucleotide substitutions, terminal additions, and/or truncations.
  • the miRNA binding site is the same length as the corresponding miRNA. In other embodiments, the miRNA binding site is one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve nucleotide(s) shorter than the corresponding miRNA at the 5' terminus, the 3' terminus, or both. In still other embodiments, the microRNA binding site is two nucleotides shorter than the corresponding microRNA at the 5' terminus, the 3' terminus, or both. The miRNA binding sites that are shorter than the corresponding miRNAs are still capable of degrading the mRNA incorporating one or more of the miRNA binding sites or preventing the mRNA from translation.
  • the miRNA binding site binds to the corresponding mature miRNA that is part of an active RISC containing Dicer. In another embodiment, binding of the miRNA binding site to the corresponding miRNA in RISC degrades the mRNA containing the miRNA binding site or prevents the mRNA from being translated. In some embodiments, the miRNA binding site has sufficient complementarity to miRNA so that a RISC complex comprising the miRNA cleaves the polynucleotide comprising the miRNA binding site. In other embodiments, the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA induces instability in the polynucleotide comprising the miRNA binding site.
  • the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA represses transcription of the polynucleotide comprising the miRNA binding site.
  • the miRNA binding site has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) from the corresponding miRNA.
  • the miRNA binding site has at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one contiguous nucleotides complementary to at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one, respectively, contiguous nucleotides of the corresponding miRNA.
  • the polynucleotide By engineering one or more miRNA binding sites into a polynucleotide of the disclosure, the polynucleotide can be targeted for degradation or reduced translation, provided the miRNA in question is available. This can reduce off-target effects upon delivery of the polynucleotide. For example, if a polynucleotide of the disclosure is not intended to be delivered to a tissue or cell but ends up there, then a miRNA abundant in the tissue or cell can inhibit the expression of the gene of interest if one or multiple binding sites of the miRNA are engineered into the 5'UTR and/or 3'UTR of the polynucleotide.
  • miRNA binding sites can be removed from polynucleotide sequences in which they naturally occur in order to increase protein expression in specific tissues.
  • a binding site for a specific miRNA can be removed from a polynucleotide to improve protein expression in tissues or cells containing the miRNA.
  • a polynucleotide of the disclosure can include at least one miRNA- binding site in the 5'UTR and/or 3'UTR in order to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells.
  • a polynucleotide of the disclosure can include two, three, four, five, six, seven, eight, nine, ten, or more miRNA-binding sites in the 5'-UTR and/or 3'-UTR in order to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells.
  • Regulation of expression in multiple tissues can be accomplished through introduction or removal of one or more miRNA binding sites.
  • the decision whether to remove or insert a miRNA binding site can be made based on miRNA expression patterns and/or their profilings in diseases. Identification of miRNAs, miRNA binding sites, and their expression patterns and role in biology have been reported (e.g., Bonauer et al., Curr Drug Targets 2010 11 :943-949; Anand and Cheresh Curr Opin Hematol 2011 18: 171- 176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec 20.
  • miRNAs and miRNA binding sites can correspond to any known sequence, including non- limiting examples described in U.S. Publication Nos. 2014/0200261, 2005/0261218, and 2005/0059005, each of which are incorporated herein by reference in their entirety.
  • tissues where miRNA are known to regulate mRNA, and thereby protein expression include, but are not limited to, liver (miR- 122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR- 17-92, miR-126), myeloid cells (miR- 142-3p, miR- 142-5p, miR- 16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR- ld, miR- 149), kidney (miR- 192, miR- 194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126).
  • liver miR- 122
  • muscle miR-133, miR-206, miR-208
  • endothelial cells miR- 17-92, miR-126
  • myeloid cells miR- 142-3p, miR- 142-5p, mi
  • microRNAs e.g. , miR- 122
  • microRNA binding site i.e. , microRNA binding site into the polynucleotides of the disclosure can effectively target the molecule for degradation or reduced translation in normal tissue (where the microRNA is abundant) while providing high levels of translation in the cancer or tumor tissue (where the microRNA is present in much lower levels). This provides a tumor-targeting approach for the methods and compositions of the disclosure.
  • miRNA binding sites include immune cell specific miRNAs including, but not limited to, hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-3p, hsa- let-7i-5p, miR- 10a-3p, miR-10a-5p, miR- 1184, hsa-let-7f-l-3p, hsa-let-7f-2-5p, hsa-let-7f-5p, miR- 125b-l-3p, miR- 125b-2-3p, miR-125b-5p, miR- 1279, miR- 130a-3p, miR-130a-5p, miR- 132- 3p, miRNAsa-let-7a-2-3p,
  • novel miRNAs can be identified in immune cell through micro-array hybridization and microtome analysis (e.g., Jima DD et al, Blood, 2010, 116:el l8-el27; Vaz C et al., BMC Genomics, 2010, 11,288, the content of each of which is incorporated herein by reference in its entirety.)
  • MiRNAs that are known to be expressed in the liver include, but are not limited to, miR- 107, miR- 122-3p, miR-122-5p, miR- 1228-3p, miR-1228-5p, miR-1249, miR- 129-5p, miR-1303, miR- 151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p, miR- 199a-3p, miR-199a-5p, miR- 199b- 3p, miR- 199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p.
  • MiRNA binding sites from any liver specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the liver.
  • Liver specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.
  • MiRNAs that are known to be expressed in the lung include, but are not limited to, let-7a-2- 3p, let-7a-3p, let-7a-5p, miR- 126-3p, miR- 126-5p, miR- 127-3p, miR- 127-5p, miR-130a-3p, miR- 130a-5p, miR-130b-3p, miR-130b-5p, miR- 133a, miR- 133b, miR- 134, miR- 18a-3p, miR-18a-5p, miR- 18b-3p, miR-18b-5p, miR-24-l-5p, miR-24-2-5p, miR-24-3p, miR-296-3p, miR-296-5p, miR- 32-3p, miR-337-3p, miR-337-5p, miR-381-3p, and miR-381-5p.
  • MiRNA binding sites from any lung specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the lung.
  • Lung specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.
  • MiRNAs that are known to be expressed in the heart include, but are not limited to, miR-1, miR- 133a, miR- 133b, miR-149-3p, miR- 149-5p, miR-186-3p, miR- 186-5p, miR-208a, miR-208b, miR-210, miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b-5p.
  • MiRNA binding sites from any heart specific micro RNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the heart.
  • Heart specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.
  • MiRNAs that are known to be expressed in the nervous system include, but are not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p, miR- 125b-l-3p, miR-125b-2-3p, miR-125b-5p,miR- 1271-3p, miR- 1271-5p, miR- 128, miR-132-5p, miR- 135a-3p, miR- 135a-5p, miR- 135b-3p, miR- 135b-5p, miR-137, miR-139-5p, miR- 139-3p, miR- 149-3p, miR-149-5p, miR- 153, miR- 181c-3p, miR- 181c-5p, miR-183-3p, miR-183-5p, miR- 190a, miR- 190b, miR-212-3p, miR-212-5p, miR-219- l-3p, miR-219-2-3p,
  • MiRNAs enriched in the nervous system further include those specifically expressed in neurons, including, but not limited to, miR- 132-3p, miR- 132-3p, miR-148b-3p, miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR- 212-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR- 328, miR-922 and those specifically expressed in glial cells, including, but not limited to, miR-1250, miR-219-l-3p, miR-219-2-3p, miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657.
  • MiRNA binding sites from any CNS specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the nervous system.
  • Nervous system specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.
  • MiRNAs that are known to be expressed in the pancreas include, but are not limited to, miR- 105-3p, miR-105-5p, miR- 184, miR- 195-3p, miR- 195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375, miR-7- l-3p, miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944.
  • MiRNA binding sites from any pancreas specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the pancreas.
  • Pancreas specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g. APC) miRNA binding sites in a polynucleotide of the disclosure.
  • MiRNAs that are known to be expressed in the kidney include, but are not limited to, miR- 122-3p, miR-145-5p, miR-17-5p, miR- 192-3p, miR- 192-5p, miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR- 30a-3p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-l-3p, miR-30c-2-3p, miR30c-5p, miR- 324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR-562.
  • MiRNA binding sites from any kidney specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the kidney.
  • Kidney specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.
  • MiRNAs that are known to be expressed in the muscle include, but are not limited to, let-7g- 3p, let-7g-5p, miR- 1, miR-1286, miR-133a, miR-133b, miR-140-3p, miR- 143-3p, miR-143-5p, miR- 145-3p, miR- 145-5p, miR- 188-3p, miR- 188-5p, miR-206, miR-208a, miR-208b, miR-25-3p, and miR-25-5p.
  • MiRNA binding sites from any muscle specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the muscle.
  • Muscle specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.
  • MiRNAs are also differentially expressed in different types of cells, such as, but not limited to, endothelial cells, epithelial cells, and adipocytes.
  • MiRNAs that are known to be expressed in endothelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p, miR- 101-3p, miR- 101-5p, miR-126-3p, miR-126-5p, miR- 1236-3p, miR- 1236-5p, miR-130a-3p, miR- 130a-5p, miR- 17-5p, miR- 17-3p, miR-18a-3p, miR- 18a-5p, miR-19a-3p, miR-19a-5p, miR- 19b- l-5p, miR-19b-2-5p, miR-19b-3p, miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p, miR-21-5p, miR-221-3p, miR-221-5p, miR-222-3p, miR-222-5p, miR-23a-3p, miR-23a
  • MiRNA binding sites from any endothelial cell specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the endothelial cells.
  • MiRNAs that are known to be expressed in epithelial cells include, but are not limited to, let- 7b-3p, let-7b-5p, miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c- 3p, miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802 and miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific in respiratory ciliated epithelial cells, let-7 family, miR-133a, miR- 133b, miR- 126 specific in lung epithelial cells, miR- 382-3p, miR-382-5p specific in renal epithelial cells, and miR-762 specific in corneal epithelial cells. MiRNA binding sites from any
  • a large group of miRNAs are enriched in embryonic stem cells, controlling stem cell self-renewal as well as the development and/or differentiation of various cell lineages, such as neural cells, cardiac, hematopoietic cells, skin cells, osteogenic cells and muscle cells (e.g., Kuppusamy KT et al., Curr. Mol Med, 2013, 13(5), 757-764; Vidigal JA and Ventura A, Semin Cancer Biol.
  • MiRNAs abundant in embryonic stem cells include, but are not limited to, let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p, miR- 103a-2-3p, miR- 103a-5p, miR-106b-3p, miR- 106b-5p, miR-1246, miR-1275, miR-138- l-3p, miR-138-2-3p, miR- 138-5p, miR-154-3p, miR- 154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p, miR- 301a-5p, miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p, miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e, miR-367-3p, miR-367-5
  • the binding sites of embryonic stem cell specific miRNAs can be included in or removed from the 3'UTR of a polynucleotide of the disclosure to modulate the development and/or differentiation of embryonic stem cells, to inhibit the senescence of stem cells in a degenerative condition (e.g. degenerative diseases), or to stimulate the senescence and apoptosis of stem cells in a disease condition (e.g. cancer stem cells).
  • a degenerative condition e.g. degenerative diseases
  • apoptosis of stem cells in a disease condition e.g. cancer stem cells.
  • MiRNA can also regulate complex biological processes such as angiogenesis (e.g., miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18: 171- 176).
  • angiogenesis e.g., miR-132
  • miRNA binding sites that are involved in such processes can be removed or introduced, in order to tailor the expression of the polynucleotides to biologically relevant cell types or relevant biological processes.
  • the polynucleotides of the disclosure are defined as auxotrophic polynucleotides.
  • the microRNA binding site (e.g. , miR-122 binding site) is fully complementary to miRNA (e.g. , miR- 122), thereby degrading the mRNA fused to the miRNA binding site.
  • the miRNA binding site is not fully complementary to the corresponding miRNA.
  • the miRNA binding site (e.g. , miR- 122 binding site) is the same length as the corresponding miRNA (e.g. , miR- 122).
  • the microRNA binding site (e.g. , miR- 122 binding site) is one nucleotide shorter than the corresponding microRNA (e.g.
  • the microRNA binding site (e.g. , miR- 122 binding site) is two nucleotides shorter than the corresponding microRNA (e.g. , miR-122) at the 5' terminus, the 3' terminus, or both.
  • the microRNA binding site (e.g. , miR-122 binding site) is three nucleotides shorter than the corresponding microRNA (e.g. , miR- 122) at the 5' terminus, the 3' terminus, or both.
  • the microRNA binding site (e.g.
  • miR-122 binding site is four nucleotides shorter than the corresponding microRNA (e.g. , miR- 122) at the 5' terminus, the 3' terminus, or both.
  • the microRNA binding site e.g. , miR-122 binding site
  • the microRNA binding site is six nucleotides shorter than the corresponding microRNA (e.g. , miR- 122) at the 5' terminus, the 3' terminus, or both.
  • the microRNA binding site (e.g. , miR- 122 binding site) is seven nucleotides shorter than the corresponding microRNA (e.g. , miR- 122) at the 5' terminus or the 3' terminus. In other embodiments, the microRNA binding site (e.g. , miR-122 binding site) is eight nucleotides shorter than the corresponding microRNA (e.g. , miR- 122) at the 5' terminus or the 3' terminus. In other embodiments, the microRNA binding site (e.g. , miR- 122 binding site) is nine nucleotides shorter than the corresponding microRNA (e.g. , miR- 122) at the 5' terminus or the 3' terminus.
  • the microRNA binding site (e.g. , miR- 122 binding site) is ten nucleotides shorter than the corresponding microRNA (e.g. , miR- 122) at the 5' terminus or the 3' terminus. In other embodiments, the microRNA binding site (e.g. , miR- 122 binding site) is eleven nucleotides shorter than the corresponding microRNA (e.g. , miR- 122) at the 5' terminus or the 3' terminus. In other embodiments, the microRNA binding site (e.g. , miR- 122 binding site) is twelve nucleotides shorter than the corresponding microRNA (e.g. , miR-122) at the 5' terminus or the 3' terminus. The miRNA binding sites that are shorter than the corresponding miRNAs are still capable of degrading the mRNA incorporating one or more of the miRNA binding sites or preventing the mRNA from translation.
  • the microRNA binding site (e.g. , miR-122 binding site) has sufficient complementarity to miRNA (e.g. , miR-122) so that a RISC complex comprising the miRNA (e.g. , miR- 122) cleaves the polynucleotide comprising the microRNA binding site.
  • the microRNA binding site (e.g. , miR-122 binding site) has imperfect complementarity so that a RISC complex comprising the miRNA (e.g. , miR- 122) induces instability in the polynucleotide comprising the microRNA binding site.
  • the microRNA binding site (e.g. , miR-122 binding site) has sufficient complementarity to miRNA (e.g. , miR-122) so that a RISC complex comprising the miRNA (e.g. , miR- 122) cleaves the polynucleotide comprising the microRNA binding site.
  • the microRNA binding site (e.g. , miR-
  • miRNA binding site (e.g. , miR- 122 binding site) has imperfect complementarity so that a RISC complex comprising the miRNA (e.g. , miR-122) represses transcription of the polynucleotide comprising the microRNA binding site.
  • the miRNA binding site (e.g. , miR- 122 binding site) has one mismatch from the corresponding miRNA (e.g. , miR- 122).
  • the miRNA binding site has two mismatches from the corresponding miRNA.
  • the miRNA binding site has three mismatches from the corresponding miRNA.
  • the miRNA binding site has four mismatches from the corresponding miRNA.
  • the miRNA binding site has five mismatches from the corresponding miRNA. In other embodiments, the miRNA binding site has six mismatches from the corresponding miRNA. In certain embodiments, the miRNA binding site has seven mismatches from the corresponding miRNA. In other embodiments, the miRNA binding site has eight mismatches from the corresponding miRNA. In other embodiments, the miRNA binding site has nine mismatches from the corresponding miRNA. In other embodiments, the miRNA binding site has ten mismatches from the corresponding miRNA. In other embodiments, the miRNA binding site has eleven mismatches from the corresponding miRNA. In other embodiments, the miRNA binding site has twelve mismatches from the corresponding miRNA.
  • the miRNA binding site (e.g. , miR-122 binding site) has at least about ten contiguous nucleotides complementary to at least about ten contiguous nucleotides of the corresponding miRNA (e.g.
  • miR- 122 miR- 122
  • at least about eleven contiguous nucleotides complementary to at least about eleven contiguous nucleotides of the corresponding miRNA at least about twelve contiguous nucleotides complementary to at least about twelve contiguous nucleotides of the corresponding miRNA, at least about thirteen contiguous nucleotides complementary to at least about thirteen contiguous nucleotides of the corresponding miRNA, or at least about fourteen contiguous nucleotides complementary to at least about fourteen contiguous nucleotides of the corresponding miRNA.
  • the miRNA binding sites have at least about fifteen contiguous nucleotides complementary to at least about fifteen contiguous nucleotides of the corresponding miRNA, at least about sixteen contiguous nucleotides complementary to at least about sixteen contiguous nucleotides of the corresponding miRNA, at least about seventeen contiguous nucleotides complementary to at least about seventeen contiguous nucleotides of the corresponding miRNA, at least about eighteen contiguous nucleotides complementary to at least about eighteen contiguous nucleotides of the corresponding miRNA, at least about nineteen contiguous nucleotides complementary to at least about nineteen contiguous nucleotides of the corresponding miRNA, at least about twenty contiguous nucleotides complementary to at least about twenty contiguous nucleotides of the corresponding miRNA, or at least about twenty one contiguous nucleotides complementary to at least about twenty one contiguous nucleotides of the corresponding miRNA.
  • the polynucleotides of the disclosure comprise at least one miR122 binding site, at least two miR122 binding sites, at least three miR122 binding sites, at least four miR122 binding sites, or at least five miR122 binding sites.
  • the miRNA binding site binds miR- 122 or is complementary to miR- 122.
  • the miRNA binding site binds to miR- 122-3p or miR- 122-5p.
  • the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 52 or 54, wherein the miRNA binding site binds to miR-122.
  • the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 54, wherein the miRNA binding site binds to miR- 122 (e.g. , with sufficient strength to induce RISC-mediated cleavage of the polynucleotide, e.g. , mRNA, comprising the miRNA binding site).
  • the miRNA binding site has less than 3 substitutions, less than 2 substitutions, or less than 1 substitution as compared to the miRNA binding site as set forth as SEQ ID NO: 54, wherein the miR binding site binds to miR-122 (e.g. , with sufficient strength to induce RISC-mediated cleavage of the polynucleotide, e.g. , mRNA, comprising the miRNA binding site).
  • a miRNA binding site (e.g. , miR-122 binding site) is inserted in the polynucleotide of the disclosure in any position of the polynucleotide (e.g. , 3' UTR); the insertion site in the polynucleotide can be anywhere in the polynucleotide as long as the insertion of the miRNA binding site in the polynucleotide does not interfere with the translation of the functional IL- 12 polypeptide or the translation of the functional membrane domain in the absence of the corresponding miRNA (e.g. , miR122); and in the presence of the miRNA (e.g.
  • miRNA binding site in the polynucleotide and the binding of the miRNA binding site to the corresponding miRNA are capable of degrading the polynucleotide or preventing the translation of the polynucleotide.
  • a miRNA binding site is inserted in a 3'UTR of the polynucleotide.
  • a miRNA binding site is inserted in at least about 30 nucleotides downstream from a stop codon in a polynucleotide of the disclosure. In other embodiments, a miRNA binding site is inserted in at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, or at least about 100 nucleotides downstream from a stop
  • a miRNA binding site is inserted in about 10 nucleotides to about 100 nucleotides, about 20 nucleotides to about 90 nucleotides, about 30 nucleotides to about 80 nucleotides, about 40 nucleotides to about 70 nucleotides, about 50 nucleotides to about 60 nucleotides, about 45 nucleotides to about 65 nucleotides downstream from the stop codon in a polynucleotide of the disclosure.
  • the miRNA binding site is inserted downstream of the stop codon in the nucleic acid sequence encoding an IL- 12 polypeptide as disclosed herein.
  • the miRNA binding site is inserted downstream of the stop codon in the nucleic acid sequence encoding membrane domain as disclosed herein. In some embodiments, the miRNA binding site is inserted downstream of the stop codon in the nucleic acid sequence encoding heterologous polypeptide as disclosed herein
  • a miRNA binding site is inserted in the polynucleotide of the disclosure in any position of the polynucleotide (e.g., the 5'UTR and/or 3'UTR).
  • the 5'UTR comprises a miRNA binding site.
  • the 3'UTR comprises a miRNA binding site.
  • the 5'UTR and the 3'UTR comprise a miRNA binding site.
  • the insertion site in the polynucleotide can be anywhere in the polynucleotide as long as the insertion of the miRNA binding site in the polynucleotide does not interfere with the translation of a functional polypeptide in the absence of the corresponding miRNA; and in the presence of the miRNA, the insertion of the miRNA binding site in the polynucleotide and the binding of the miRNA binding site to the corresponding miRNA are capable of degrading the polynucleotide or preventing the translation of the polynucleotide.
  • a miRNA binding site is inserted in at least about 30 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the disclosure comprising the ORF. In some embodiments, a miRNA binding site is inserted in at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, or at least about 100 nucle
  • a miRNA binding site is inserted in about 10 nucleotides to about 100 nucleotides, about 20 nucleotides to about 90 nucleotides, about 30 nucleotides to about 80 nucleotides, about 40 nucleotides to about 70 nucleotides, about 50 nucleotides to about 60 nucleotides, about 45 nucleotides to about 65 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the disclosure.
  • MiRNA gene regulation can be influenced by the sequence surrounding the miRNA such as, but not limited to, the species of the surrounding sequence, the type of sequence (e.g., heterologous, homologous, exogenous, endogenous, or artificial), regulatory elements in the surrounding sequence and/or structural elements in the surrounding sequence.
  • the miRNA can be influenced by the 5 'UTR and/or 3 'UTR.
  • a non-human 3 'UTR can increase the regulatory effect of the miRNA sequence on the expression of a polypeptide of interest compared to a human 3 'UTR of the same sequence type.
  • other regulatory elements and/or structural elements of the 5 'UTR can influence miRNA mediated gene regulation.
  • a regulatory element and/or structural element is a structured IRES (Internal Ribosome Entry Site) in the 5 'UTR, which is necessary for the binding of translational elongation factors to initiate protein translation. EIF4A2 binding to this secondarily structured element in the 5'-UTR is necessary for miRNA mediated gene expression (Meijer HA et ah, Science, 2013, 340, 82-85, herein incorporated by reference in its entirety).
  • the polynucleotides of the disclosure can further include this structured 5 'UTR in order to enhance microRNA mediated gene regulation.
  • At least one miRNA binding site can be engineered into the 3 'UTR of a polynucleotide of the disclosure.
  • at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more miRNA binding sites can be engineered into a 3 'UTR of a polynucleotide of the disclosure.
  • 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1 miRNA binding sites can be engineered into the 3 'UTR of a polynucleotide of the disclosure.
  • miRNA binding sites incorporated into a polynucleotide of the disclosure can be the same or can be different miRNA sites.
  • a combination of different miRNA binding sites incorporated into a polynucleotide of the disclosure can include combinations in which more than one copy of any of the different miRNA sites are incorporated.
  • miRNA binding sites incorporated into a polynucleotide of the disclosure can target the same or different tissues in the body.
  • tissue-, cell- type-, or disease- specific miRNA binding sites in the 3'-UTR of a polynucleotide of the disclosure can be reduced.
  • a miRNA binding site can be engineered near the 5' terminus of the 3 'UTR, about halfway between the 5' terminus and 3' terminus of the 3 'UTR and/or near the 3' terminus of the 3 'UTR in a polynucleotide of the disclosure.
  • a miRNA binding site can be engineered near the 5' terminus of the 3 'UTR and about halfway between the 5' terminus and 3' terminus of the 3 'UTR.
  • a miRNA binding site can be engineered near the 3' terminus of the 3'UTR and about halfway between the 5' terminus and 3' terminus of the 3'UTR.
  • a miRNA binding site can be engineered near the 5' terminus of the 3'UTR and near the 3' terminus of the 3'UTR.
  • a 3'UTR can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA binding sites.
  • the miRNA binding sites can be complementary to a miRNA, miRNA seed sequence, and/or miRNA sequences flanking the seed sequence.
  • a polynucleotide of the disclosure can be engineered to include more than one miRNA site expressed in different tissues or different cell types of a subject.
  • a polynucleotide of the disclosure can be engineered to include miR-192 and miR- 122 to regulate expression of the polynucleotide in the liver and kidneys of a subject.
  • a polynucleotide of the disclosure can be engineered to include more than one miRNA site for the same tissue.
  • the therapeutic window and or differential expression associated with the polypeptide encoded by a polynucleotide of the disclosure can be altered with a miRNA binding site.
  • a polynucleotide encoding a polypeptide that provides a death signal can be designed to be more highly expressed in cancer cells by virtue of the miRNA signature of those cells.
  • the polynucleotide encoding the binding site for that miRNA (or miRNAs) would be more highly expressed.
  • the polypeptide that provides a death signal triggers or induces cell death in the cancer cell.
  • Neighboring non-cancer cells, harboring a higher expression of the same miRNA would be less affected by the encoded death signal as the polynucleotide would be expressed at a lower level due to the effects of the miRNA binding to the binding site or "sensor" encoded in the 3'UTR.
  • cell survival or cytoprotective signals can be delivered to tissues containing cancer and non-cancerous cells where a miRNA has a higher expression in the cancer cells— the result being a lower survival signal to the cancer cell and a larger survival signal to the normal cell.
  • Multiple polynucleotides can be designed and administered having different signals based on the use of miRNA binding sites as described herein.
  • the expression of a polynucleotide of the disclosure can be controlled by incorporating at least one sensor sequence in the polynucleotide and formulating the polynucleotide for administration.
  • a polynucleotide of the disclosure can be targeted to a tissue or cell by incorporating a miRNA binding site and formulating the polynucleotide in a lipid nanoparticle comprising a cationic lipid, including any of the lipids described herein.
  • a polynucleotide of the disclosure can be engineered for more targeted expression in specific tissues, cell types, or biological conditions based on the expression patterns of miRNAs in the different tissues, cell types, or biological conditions. Through introduction of tissue-specific miRNA binding sites, a polynucleotide of the disclosure can be designed for optimal protein expression in a tissue or cell, or in the context of a biological condition.
  • a polynucleotide of the disclosure can be designed to incorporate miRNA binding sites that either have 100% identity to known miRNA seed sequences or have less than 100% identity to miRNA seed sequences.
  • a polynucleotide of the disclosure can be designed to incorporate miRNA binding sites that have at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to known miRNA seed sequences.
  • the miRNA seed sequence can be partially mutated to decrease miRNA binding affinity and as such result in reduced down modulation of the polynucleotide.
  • the degree of match or mismatch between the miRNA binding site and the miRNA seed can act as a rheostat to more finely tune the ability of the miRNA to modulate protein expression.
  • mutation in the non-seed region of a miRNA binding site can also impact the ability of a miRNA to modulate protein expression.
  • a miRNA sequence can be incorporated into the loop of a stem loop.
  • a miRNA seed sequence can be incorporated in the loop of a stem loop and a miRNA binding site can be incorporated into the 5 Or 3 ' stem of the stem loop.
  • a translation enhancer element can be incorporated on the 5 'end of the stem of a stem loop and a miRNA seed can be incorporated into the stem of the stem loop.
  • a TEE can be incorporated on the 5' end of the stem of a stem loop, a miRNA seed can be incorporated into the stem of the stem loop and a miRNA binding site can be incorporated into the 3 ' end of the stem or the sequence after the stem loop.
  • the miRNA seed and the miRNA binding site can be for the same and/or different miRNA sequences.
  • the incorporation of a miRNA sequence and/or a TEE sequence changes the shape of the stem loop region which can increase and/or decrease translation, (see e.g., Kedde et al., "A Pumilio-induced RNA structure switch in p27-3'UTR controls miR-221 and miR-22 accessibility.” Nature Cell Biology. 2010, incorporated herein by reference in its entirety).
  • the 5'-UTR of a polynucleotide of the disclosure can comprise at least one miRNA sequence.
  • the miRNA sequence can be, but is not limited to, a 19 or 22 nucleotide sequence and/or a miRNA sequence without the seed.
  • the miRNA sequence in the 5'UTR can be used to stabilize a polynucleotide of the disclosure described herein.
  • a miRNA sequence in the 5'UTR of a polynucleotide of the disclosure can be used to decrease the accessibility of the site of translation initiation such as, but not limited to a start codon. See, e.g., Matsuda et al., PLoS One. 2010 l l(5):el5057; incorporated herein by reference in its entirety, which used antisense locked nucleic acid (LNA) oligonucleotides and exon-junction complexes (EJCs) around a start codon (-4 to +37 where the A of the AUG codons is +1) in order to decrease the accessibility to the first start codon (AUG).
  • LNA antisense locked nucleic acid
  • EJCs exon-junction complexes
  • a polynucleotide of the disclosure can comprise a miRNA sequence, instead of the LNA or EJC sequence described by Matsuda et al, near the site of translation initiation in order to decrease the accessibility to the site of translation initiation.
  • the site of translation initiation can be prior to, after or within the miRNA sequence.
  • the site of translation initiation can be located within a miRNA sequence such as a seed sequence or binding site.
  • the site of translation initiation may be located within a miR-122 sequence such as the seed sequence or the mir- 122 binding site.
  • a polynucleotide of the disclosure can include at least one miRNA in order to dampen expression of the encoded polypeptide in a tissue or cell of interest.
  • a polynucleotide of the disclosure can include at least one miR-122 binding site in order to dampen expression of an encoded polypeptide of interest in the liver.
  • a polynucleotide of the disclosure can include at least one miR-142-3p binding site, miR- 142-3p seed sequence, miR- 142-3p binding site without the seed, miR-142-5p binding site, miR- 142-5p seed sequence, miR-142-5p binding site without the seed, miR-146 binding site, miR- 146 seed sequence and/or miR-146 binding site without the seed sequence.
  • a polynucleotide of the disclosure can comprise at least one miRNA binding site in the 3 'UTR in order to selectively degrade mRNA therapeutics in the immune cells to subdue unwanted immunogenic reactions caused by therapeutic delivery.
  • the miRNA binding site can make a polynucleotide of the disclosure more unstable in antigen presenting cells.
  • these miRNAs include mir- 122-5p or mir- 122-3p.
  • a polynucleotide of the disclosure comprises at least one miRNA sequence in a region of the polynucleotide that can interact with a RNA binding protein.
  • the polynucleotide of the disclosure (e.g., a RNA, e.g., an mRNA) comprises (i) a sequence-optimized nucleotide sequence encoding an IL- 12 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), (ii) a sequence-optimized nucleotide sequence encoding a membrane domain, and (iii) a miRNA binding site (e.g., a miRNA binding site that binds to miR- 122).
  • a sequence-optimized nucleotide sequence encoding an IL- 12 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), (ii) a sequence-optimized nucleotide sequence encoding a membrane domain, and (iii) a miRNA binding site (e.g., a miRNA binding site that binds to miR- 122).
  • the polynucleotide of the disclosure comprises a nucleobase-modified sequence and a miRNA binding site disclosed herein, e.g., a miRNA binding site that binds to miR- 122.
  • the polynucleotides of the disclosure comprising a miRNA binding site are formulated with a delivery agent, e.g., a compound having the Formula (I).
  • a polynucleotide of the present disclosure further comprises a 3'
  • 3'-UTR is the section of mRNA that immediately follows the translation termination codon and often contains regulatory regions that post-transcriptionally influence gene expression. Regulatory regions within the 3'-UTR can influence polyadenylation, translation efficiency, localization, and stability of the mRNA.
  • the 3'-UTR useful for the disclosure comprises a binding site for regulatory proteins or microRNAs.
  • the 3' UTR useful for the polynucleotides of the disclosure comprises a 3'UTR selected from those shown in this application (e.g., SEQ ID NOs: 222-224). In some embodiments, the 3'UTR useful for the polynucleotides of the disclosure comprises a 3'UTR comprising the sequence set forth in SEQ ID NO: 283.
  • the 3' UTR sequence useful for the disclosure comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of 3'UTR sequences listed herein and any combination thereof. 17. Regions having a 5' Cap
  • the disclosure also includes a polynucleotide that comprises both a 5' Cap and a polynucleotide of the present disclosure.
  • the 5' cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species.
  • CBP mRNA Cap Binding Protein
  • the cap further assists the removal of 5' proximal introns during mRNA splicing.
  • Endogenous mRNA molecules can be 5 '-end capped generating a 5 '-ppp-5 '-triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule.
  • This 5 '-guanylate cap can then be methylated to generate an N7-methyl- guanylate residue.
  • the ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5' end of the mRNA can optionally also be 2'-0-methylated.
  • 5'-decapping through hydrolysis and cleavage of the guanylate cap structure can target a nucleic acid molecule, such as an mRNA molecule, for degradation.
  • the polynucleotides of the present disclosure incorporate a cap moiety.
  • polynucleotides of the present disclosure comprise a non- hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5 '-ppp-5' phosphorodiester linkages, modified nucleotides can be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) can be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5 '-ppp-5' cap. Additional modified guanosine nucleotides can be used such as a-methyl-phosphonate and seleno -phosphate nucleotides.
  • Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5 '-terminal and/or 5 '-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2'-hydroxyl group of the sugar ring.
  • Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as a polynucleotide that functions as an mRNA molecule.
  • Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5 '-caps in their chemical structure, while retaining cap function.
  • Cap analogs can be chemically (i.e. , non-enzymatically) or enzymatically synthesized and/or linked to the polynucleotides of the disclosure.
  • the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3 '-0-methyl group (i.e. , N7,3 '-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m 7 G-3 'mppp-G; which may equivalently be designated 3 ' 0-Me-m7G(5')ppp(5')G).
  • the 3 '-0 atom of the other, unmodified, guanine becomes linked to the 5 '-terminal nucleotide of the capped polynucleotide.
  • the N7- and 3 '- O-methlyated guanine provides the terminal moiety of the capped polynucleotide.
  • mCAP which is similar to ARCA but has a 2'-0-methyl group on guanosine (i.e. , N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7 Gm-ppp-G).
  • the cap is a dinucleotide cap analog.
  • the dinucleotide cap analog can be modified at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in U.S. Patent No. US 8,519,110, the contents of which are herein incorporated by reference in its entirety.
  • the cap is a cap analog is a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog known in the art and/or described herein.
  • Non-limiting examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4- chlorophenoxyethyl)-G(5')ppp(5')G and a N7-(4-chlorophenoxyethyl)-m 3 ⁇ °G(5')ppp(5')G cap analog (See, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al.
  • a cap analog of the present disclosure is a 4-chloro/bromophenoxyethyl analog.
  • cap analogs allow for the concomitant capping of a polynucleotide or a region thereof, in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 '-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, can lead to reduced translational competency and reduced cellular stability.
  • Polynucleotides of the disclosure can also be capped post-manufacture (whether IVT or chemical synthesis), using enzymes, in order to generate more authentic 5'-cap structures.
  • the phrase "more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects.
  • Non- limiting examples of more authentic 5 'cap structures of the present disclosure are those that, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5'decapping, as compared to synthetic 5 'cap structures known in the art (or to a wild-type, natural or physiological 5 'cap structure).
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2 '-O-methyltransferase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5 '-terminal nucleotide of a polynucleotide and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5'- terminal nucleotide of the mRNA contains a 2'-0-methyl.
  • Capl structure Such a structure is termed the Capl structure.
  • Cap structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), and 7mG(5')-ppp(5')NlmpN2mp (cap 2).
  • capping chimeric polynucleotides post-manufacture can be more efficient as nearly 100% of the chimeric polynucleotides can be capped. This is in contrast to -80% when a cap analog is linked to a chimeric polynucleotide in the course of an in vitro transcription reaction.
  • 5' terminal caps can include endogenous caps or cap analogs.
  • a 5' terminal cap can comprise a guanine analog.
  • Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido- guanosine.
  • the polynucleotides of the present disclosure further comprise a poly- A tail.
  • terminal groups on the poly-A tail can be incorporated for stabilization.
  • a poly-A tail comprises des-3' hydroxyl tails.
  • a long chain of adenine nucleotides can be added to a polynucleotide such as an mRNA molecule in order to increase stability.
  • the 3' end of the transcript can be cleaved to free a 3' hydroxyl.
  • poly-A polymerase adds a chain of adenine nucleotides to the RNA.
  • polyadenylation adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long.
  • PolyA tails can also be added after the construct is exported from the nucleus.
  • terminal groups on the polyA tail can be incorporated for stabilization.
  • Polynucleotides of the present disclosure can include des-3' hydroxyl tails. They can also include structural moieties or 2'-Omethyl modifications as taught by Junjie Li, et al. (Current Biology, Vol. 15, 1501-1507, August 23, 2005, the contents of which are incorporated herein by reference in its entirety).
  • the polynucleotides of the present disclosure can be designed to encode transcripts with alternative polyA tail structures including histone mRNA. According to Norbury, "Terminal uridylation has also been detected on human replication-dependent histone mRNAs. The turnover of these mRNAs is thought to be important for the prevention of potentially toxic histone accumulation following the completion or inhibition of chromosomal DNA replication.
  • mRNAs are distinguished by their lack of a 3 ' poly(A) tail, the function of which is instead assumed by a stable stem-loop structure and its cognate stem-loop binding protein (SLBP); the latter carries out the same functions as those of PABP on polyadenylated mRNAs" (Norbury, "Cytoplasmic RNA: a case of the tail wagging the dog," Nature Reviews Molecular Cell Biology; AOP, published online 29 August 2013; doi: 10.1038/nrm3645) the contents of which are incorporated herein by reference in its entirety.
  • SLBP stem-loop binding protein
  • the length of a poly-A tail when present, is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides).
  • the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,
  • the polynucleotide or region thereof includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from from about 30 to
  • the poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design can be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate product expressed from the polynucleotides.
  • the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof.
  • the poly-A tail can also be designed as a fraction of the polynucleotides to which it belongs.
  • the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A tail.
  • engineered binding sites and conjugation of polynucleotides for Poly-A binding protein can enhance expression.
  • multiple distinct polynucleotides can be linked together via the PABP (Poly-A binding protein) through the 3 '-end using modified nucleotides at the 3 '-terminus of the poly-A tail.
  • Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72hr and day 7 post-transfection.
  • the polynucleotides of the present disclosure are designed to include a polyA-G quartet region.
  • the G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA.
  • the G-quartet is incorporated at the end of the poly-A tail.
  • the resultant polynucleotide is assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the polyA-G quartet results in protein production from an mRNA equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone. 19. Start codon region
  • the disclosure also includes a polynucleotide that comprises both a start codon region and the polynucleotide described herein.
  • the polynucleotides of the present disclosure can have regions that are analogous to or function like a start codon region.
  • the translation of a polynucleotide can initiate on a codon that is not the start codon AUG.
  • Translation of the polynucleotide can initiate on an alternative start codon such as, but not limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, TTG/UUG (see Touriol et al. Biology of the Cell 95 (2003) 169- 178 and Matsuda and Mauro PLoS ONE, 2010 5: 11 ; the contents of each of which are herein incorporated by reference in its entirety).
  • the translation of a polynucleotide begins on the alternative start codon ACG.
  • polynucleotide translation begins on the alternative start codon CTG or CUG.
  • the translation of a polynucleotide begins on the alternative start codon GTG or GUG.
  • Nucleotides flanking a codon that initiates translation such as, but not limited to, a start codon or an alternative start codon, are known to affect the translation efficiency, the length and/or the structure of the polynucleotide. (See, e.g., Matsuda and Mauro PLoS ONE, 2010 5: 11 ; the contents of which are herein incorporated by reference in its entirety). Masking any of the nucleotides flanking a codon that initiates translation can be used to alter the position of translation initiation, translation efficiency, length and/or structure of a polynucleotide.
  • a masking agent can be used near the start codon or alternative start codon in order to mask or hide the codon to reduce the probability of translation initiation at the masked start codon or alternative start codon.
  • masking agents include antisense locked nucleic acids (LNA) polynucleotides and exon-junction complexes (EJCs) (See, e.g., Matsuda and Mauro describing masking agents LNA polynucleotides and EJCs (PLoS ONE, 2010 5: 11); the contents of which are herein incorporated by reference in its entirety).
  • a masking agent can be used to mask a start codon of a polynucleotide in order to increase the likelihood that translation will initiate on an alternative start codon.
  • a masking agent can be used to mask a first start codon or alternative start codon in order to increase the chance that translation will initiate on a start codon or alternative start codon downstream to the masked start codon or alternative start codon.
  • a start codon or alternative start codon can be located within a perfect complement for a miR binding site. The perfect complement of a miR binding site can help control the translation, length and/or structure of the polynucleotide similar to a masking agent.
  • the start codon or alternative start codon can be located in the middle of a perfect complement for a miRNA binding site.
  • the start codon or alternative start codon can be located after the first nucleotide, second nucleotide, third nucleotide, fourth nucleotide, fifth nucleotide, sixth nucleotide, seventh nucleotide, eighth nucleotide, ninth nucleotide, tenth nucleotide, eleventh nucleotide, twelfth nucleotide, thirteenth nucleotide, fourteenth nucleotide, fifteenth nucleotide, sixteenth nucleotide, seventeenth nucleotide, eighteenth nucleotide, nineteenth nucleotide, twentieth nucleotide or twenty-first nucleotide.
  • the start codon of a polynucleotide can be removed from the polynucleotide sequence in order to have the translation of the polynucleotide begin on a codon that is not the start codon.
  • Translation of the polynucleotide can begin on the codon following the removed start codon or on a downstream start codon or an alternative start codon.
  • the start codon ATG or AUG is removed as the first 3 nucleotides of the polynucleotide sequence in order to have translation initiate on a downstream start codon or alternative start codon.
  • the polynucleotide sequence where the start codon was removed can further comprise at least one masking agent for the downstream start codon and/or alternative start codons in order to control or attempt to control the initiation of translation, the length of the polynucleotide and/or the structure of the polynucleotide.
  • the disclosure also includes a polynucleotide that comprises both a stop codon region and the nucleic acid sequences described herein.
  • the polynucleotides of the present disclosure can include at least two stop codons before the 3' untranslated region (UTR).
  • the stop codon can be selected from TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA.
  • the polynucleotides of the present disclosure include the stop codon TGA in the case or DNA, or the stop codon UGA in the case of RNA, and one additional stop codon.
  • the addition stop codon can be TAA or UAA.
  • the polynucleotides of the present disclosure include three consecutive stop codons, four stop codons, or more. 21. Insertions and Substitutions
  • the disclosure also includes a polynucleotide of the present disclosure that further comprises insertions and/or substitutions.
  • the 5'UTR of the polynucleotide can be replaced by the insertion of at least one region and/or string of nucleosides of the same base.
  • the region and/or string of nucleotides can include, but is not limited to, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 nucleotides and the nucleotides can be natural and/or unnatural.
  • the group of nucleotides can include 5-8 adenine, cytosine, thymine, a string of any of the other nucleotides disclosed herein and/or combinations thereof.
  • the 5'UTR of the polynucleotide can be replaced by the insertion of at least two regions and/or strings of nucleotides of two different bases such as, but not limited to, adenine, cytosine, thymine, any of the other nucleotides disclosed herein and/or combinations thereof.
  • the 5'UTR can be replaced by inserting 5-8 adenine bases followed by the insertion of 5-8 cytosine bases.
  • the 5'UTR can be replaced by inserting 5-8 cytosine bases followed by the insertion of 5-8 adenine bases.
  • the polynucleotide can include at least one substitution and/or insertion downstream of the transcription start site that can be recognized by an RNA polymerase.
  • at least one substitution and/or insertion can occur downstream of the transcription start site by substituting at least one nucleic acid in the region just downstream of the transcription start site (such as, but not limited to, +1 to +6).
  • NTP nucleotide triphosphate
  • the polynucleotide can include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or at least 13 guanine bases downstream of the transcription start site.
  • the polynucleotide can include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 guanine bases in the region just downstream of the transcription start site.
  • the guanine bases can be substituted by at least 1, at least 2, at least 3 or at least 4 adenine nucleotides.
  • the guanine bases can be substituted by at least 1, at least 2, at least 3 or at least 4 cytosine bases.
  • the guanine bases in the region are GGGAGA the guanine bases can be substituted by at least 1, at least 2, at least 3 or at least 4 thymine, and/or any of the nucleotides described herein.
  • the polynucleotide can include at least one substitution and/or insertion upstream of the start codon.
  • the start codon is the first codon of the protein coding region whereas the transcription start site is the site where transcription begins.
  • the polynucleotide can include, but is not limited to, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 substitutions and/or insertions of nucleotide bases.
  • the nucleotide bases can be inserted or substituted at 1, at least 1, at least 2, at least 3, at least 4 or at least 5 locations upstream of the start codon.
  • the nucleotides inserted and/or substituted can be the same base (e.g., all A or all C or all T or all G), two different bases (e.g., A and C, A and T, or C and T), three different bases (e.g., A, C and T or A, C and T) or at least four different bases.
  • the guanine base upstream of the coding region in the polynucleotide can be substituted with adenine, cytosine, thymine, or any of the nucleotides described herein.
  • the substitution of guanine bases in the polynucleotide can be designed so as to leave one guanine base in the region downstream of the transcription start site and before the start codon (see Esvelt et al. Nature (2011) 472(7344):499-503; the contents of which is herein incorporated by reference in its entirety).
  • at least 5 nucleotides can be inserted at 1 location downstream of the transcription start site but upstream of the start codon and the at least 5 nucleotides can be the same base type.
  • a polynucleotide of the present disclosure for example a
  • polynucleotide comprising an mRNA nucleotide sequence encoding a tethered IL-12 polypeptide comprises from 5' to 3' end:
  • a 5' UTR such as the sequences provided above, comprising a 5' cap provided above;
  • a nucleic acid sequence encoding an IL-12 polypeptide disclosed herein e.g. , a nucleic acid sequence encoding IL- 12B, a nucleic acid sequence encoding IL- 12A, and, optionally, a nucleic acid sequence encoding a linker as disclosed herein that connects IL- 12B and IL- 12A, e.g., a sequence optimized nucleic acid sequence encoding an IL-12 polypeptide disclosed herein;
  • a polynucleotide of the present disclosure for example a
  • polynucleotide comprising an mRNA nucleotide sequence encoding a tethered IL-12 polypeptide comprises from 5' to 3' end:
  • a 5' UTR such as the sequences provided above, comprising a 5' cap
  • nucleic acid sequence encoding a linker as disclosed herein that connects the membrane domain as disclosed herein to an IL-12 polypeptide as disclosed herein;
  • nucleic acid sequence encoding an IL-12 polypeptide disclosed herein e.g. , a nucleic acid sequence encoding IL- 12B, a nucleic acid sequence encoding IL- 12A, and, optionally, a nucleic acid sequence encoding a linker as disclosed herein that connects IL- 12B and IL- 12A, e.g., a sequence optimized nucleic acid sequence encoding an IL-12 polypeptide disclosed herein;
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miRNA-122.
  • the 5'UTR comprises the miRNA binding site.
  • a polynucleotide of the present disclosure comprises a nucleotide sequence encoding a polypeptide sequence at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% identical to the protein sequence of a wild type IL- 12 (e.g., isoform 1, 2, 3, or 4).
  • a wild type IL- 12 e.g., isoform 1, 2, 3, or 4
  • the present disclosure also provides methods for making a polynucleotide of the disclosure or a complement thereof.
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • a polynucleotide disclosed herein can be constructed using in vitro transcription.
  • a polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein can be constructed by chemical synthesis using an oligonucleotide synthesizer.
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • a host cell is a eukaryotic cell, e.g., in vitro mammalian cells.
  • Naturally occurring nucleosides, non-naturally occurring nucleosides, or combinations thereof, can totally or partially naturally replace occurring nucleosides present in the candidate nucleotide sequence and can be incorporated into a sequence-optimized nucleotide sequence (e.g., a RNA, e.g., an mRNA) as disclosed herein.
  • a sequence-optimized nucleotide sequence e.g., a RNA, e.g., an mRNA
  • the resultant polynucleotides, e.g., mRNAs can then be examined for their ability to produce protein and/or produce a therapeutic outcome.
  • the polynucleotides of the present disclosure disclosed herein can be transcribed using an in vitro transcription (IVT) system.
  • the system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.
  • NTPs can be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs.
  • the polymerase can be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to incorporate polynucleotides disclosed herein. See U.S. Publ. No. US20130259923, which is herein incorporated by reference in its entirety.
  • RNA polymerases can be modified by inserting or deleting amino acids of the RNA polymerase sequence.
  • the RNA polymerase can be modified to exhibit an increased ability to incorporate a 2 '-modified nucleotide triphosphate compared to an unmodified RNA polymerase (see International Publication WO2008078180 and U.S. Patent 8,101,385; herein incorporated by reference in their entireties).
  • Variants can be obtained by evolving an RNA polymerase, optimizing the RNA polymerase amino acid and/or nucleic acid sequence and/or by using other methods known in the art.
  • T7 RNA polymerase variants can be evolved using the continuous directed evolution system set out by Esvelt et al.
  • T7 RNA polymerase can encode at least one mutation such as, but not limited to, lysine at position 93 substituted for threonine (K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S 128R, A136T, N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A, Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P, A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A, H523L, H
  • T7 RNA polymerase variants can encode at least mutation as described in U.S. Pub. Nos. 20100120024 and 20070117112; herein incorporated by reference in their entireties.
  • Variants of RNA polymerase can also include, but are not limited to, substitutional variants, conservative amino acid substitution, insertional variants, deletional variants and/or covalent derivatives.
  • the polynucleotide can be designed to be recognized by the wild type or variant RNA polymerases. In doing so, the polynucleotide can be modified to contain sites or regions of sequence changes from the wild type or parent chimeric polynucleotide.
  • Polynucleotide or nucleic acid synthesis reactions can be carried out by enzymatic methods utilizing polymerases.
  • Polymerases catalyze the creation of phosphodiester bonds between nucleotides in a polynucleotide or nucleic acid chain.
  • DNA polymerases can be divided into different families based on amino acid sequence comparison and crystal structure analysis.
  • DNA polymerase I poly I
  • a polymerase family including the Klenow fragments of E. coli, Bacillus DNA polymerase I, Thermus aquaticus (Taq) DNA polymerases, and the T7 RNA and DNA polymerases, is among the best studied of these families.
  • DNA polymerase a or B polymerase family, including all eukaryotic replicating DNA polymerases and polymerases from phages T4 and RB69. Although they employ similar catalytic mechanism, these families of polymerases differ in substrate specificity, substrate analog-incorporating efficiency, degree and rate for primer extension, mode of DNA synthesis, exonuclease activity, and sensitivity against inhibitors.
  • DNA polymerases are also selected based on the optimum reaction conditions they require, such as reaction temperature, pH, and template and primer concentrations. Sometimes a combination of more than one DNA polymerases is employed to achieve the desired DNA fragment size and synthesis efficiency. For example, Cheng et al. increase pH, add glycerol and dimethyl sulfoxide, decrease denaturation times, increase extension times, and utilize a secondary thermostable DNA polymerase that possesses a ⁇ to 5' exonuclease activity to effectively amplify long targets from cloned inserts and human genomic DNA. (Cheng et al., PNAS 91:5695-5699 (1994), the contents of which are incorporated herein by reference in their entirety).
  • RNA polymerases from bacteriophage T3, T7, and SP6 have been widely used to prepare RNAs for biochemical and biophysical studies.
  • RNA polymerases, capping enzymes, and poly-A polymerases are disclosed in the co-pending International Publication No. WO2014028429, the contents of which are incorporated herein by reference in their entirety.
  • the RNA polymerase which can be used in the synthesis of the polynucleotides of the present disclosure is a Syn5 RNA polymerase, (see Zhu et al. Nucleic Acids Research 2013, doi: 10.1093/nar/gktl l93, which is herein incorporated by reference in its entirety).
  • the Syn5 RNA polymerase was recently characterized from marine cyanophage Syn5 by Zhu et al. where they also identified the promoter sequence (see Zhu et al. Nucleic Acids Research 2013, the contents of which is herein incorporated by reference in its entirety). Zhu et al.
  • Syn5 RNA polymerase catalyzed RNA synthesis over a wider range of temperatures and salinity as compared to T7 RNA polymerase. Additionally, the requirement for the initiating nucleotide at the promoter was found to be less stringent for Syn5 RNA polymerase as compared to the T7 RNA polymerase making Syn5 RNA polymerase promising for RNA synthesis.
  • a Syn5 RNA polymerase can be used in the synthesis of the polynucleotides described herein. As a non-limiting example, a Syn5 RNA polymerase can be used in the synthesis of the polynucleotide requiring a precise 3 '-terminus.
  • a Syn5 promoter can be used in the synthesis of the polynucleotides.
  • the Syn5 promoter can be 5 '-ATTGGGCACCCGTAAGGG-3 ' (SEQ ID NO: 47) as described by Zhu et al. (Nucleic Acids Research 2013).
  • a Syn5 RNA polymerase can be used in the synthesis of polynucleotides comprising at least one chemical modification described herein and/or known in the art (see e.g., the incorporation of pseudo-UTP and 5Me-CTP described in Zhu et al. Nucleic Acids Research 2013).
  • the polynucleotides described herein can be synthesized using a Syn5 RNA polymerase which has been purified using modified and improved purification procedure described by Zhu et al. (Nucleic Acids Research 2013).
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence-based amplification
  • TMA transcription mediated amplification
  • RCA rolling-circle amplification
  • Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest, such as a polynucleotide of the disclosure.
  • an isolated polypeptide of interest such as a polynucleotide of the disclosure.
  • a single DNA or RNA oligomer containing a codon-optimized nucleotide sequence coding for the particular isolated polypeptide can be synthesized.
  • several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated.
  • the individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • a polynucleotide disclosed herein e.g., a RNA, e.g., an mRNA
  • a RNA e.g., an mRNA
  • Purification of the polynucleotides described herein can include, but is not limited to, polynucleotide clean-up, quality assurance and quality control. Clean-up can be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc., Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
  • AGENCOURT® beads Beckman Coulter Genomics, Danvers, MA
  • poly-T beads poly-T beads
  • LNATM oligo-T capture probes EXIQON® Inc., Vedbaek, Denmark
  • HPLC based purification methods such as, but not limited to, strong anion exchange HPLC,
  • purified when used in relation to a polynucleotide such as a “purified polynucleotide” refers to one that is separated from at least one contaminant.
  • a "contaminant” is any substance that makes another unfit, impure or inferior.
  • a purified polynucleotide e.g., DNA and RNA
  • purification of a polynucleotide of the disclosure removes impurities that can reduce or remove an unwanted immune response, e.g., reducing cytokine activity.
  • the polynucleotide of the disclosure is purified prior to administration using column chromatography (e.g. , strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), or (LCMS)).
  • column chromatography e.g. , strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), or (LCMS)
  • the polynucleotide of the disclosure purified using column
  • chromatography presents increased expression of the encoded IL- 12 protein compared to the expression level obtained with the same polynucleotide of the present disclosure purified by a different purification method.
  • a column chromatography e.g.
  • purified polynucleotide comprises a nucleotide sequence encoding an IL- 12 polypeptide comprising one or more of the point mutations known in the art.
  • the use of RP-HPLC purified polynucleotide increases IL- 12 protein expression levels in cells when introduced into those cells, e.g., by 10- 100%, i.e., at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% with respect to the expression levels of IL- 12 protein in the cells before the RP- HPLC purified polynucleotide was introduced in the cells, or after a non-RP-HPLC purified polynucleotide was introduced in the cells.
  • the use of RP-HPLC purified polynucleotide increases functional IL- 12 protein expression levels in cells when introduced into those cells, e.g., by 10-100%, i.e., at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% with respect to the functional expression levels of IL- 12 protein in the cells before the RP-HPLC purified polynucleotide was introduced in the cells, or after a non-RP- HPLC purified polynucleotide was introduced in the cells.
  • the use of RP-HPLC purified polynucleotide increases detectable IL- 12 activity in cells when introduced into those cells, e.g., by 10- 100%, i.e., at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% with respect to the activity levels of functional IL-12 in the cells before the RP- HPLC purified polynucleotide was introduced in the cells, or after a non-RP-HPLC purified polynucleotide was introduced in the cells.
  • the purified polynucleotide is at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, at least about 96% pure, at least about 97% pure, at least about 98% pure, at least about 99% pure, or about 100% pure.
  • a quality assurance and/or quality control check can be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.
  • the polynucleotide can be sequenced by methods including, but not limited to reverse-transcriptase- PCR. d. Quantification of Expressed Polynucleotides Encoding Tethered IL-12 Polypeptides
  • the polynucleotides of the present disclosure can be quantified according to methods known in the art.
  • the polynucleotides of the present disclosure can be quantified in exosomes or when derived from one or more bodily fluid.
  • bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood.
  • exosomes can be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.
  • the exosome quantification method a sample of not more than 2mL is obtained from the subject and the exosomes isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • the level or concentration of a polynucleotide can be an expression level, presence, absence, truncation or alteration of the administered construct. It is advantageous to correlate the level with one or more clinical phenotypes or with an assay for a human disease biomarker.
  • the assay can be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes can be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods.
  • Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • the polynucleotide can be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).
  • UV/Vis ultraviolet visible spectroscopy
  • a non-limiting example of a UV/Vis spectrometer is a NANODROP ® spectrometer (ThermoFisher, Waltham, MA).
  • NANODROP ® spectrometer ThermoFisher, Waltham, MA.
  • Degradation of the polynucleotide can be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
  • HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
  • compositions and formulations that comprise any of the polynucleotides described above.
  • the composition or formulation further comprises a delivery agent.
  • the composition or formulation can contain a polynucleotide comprising a sequence optimized nucleic acid sequence disclosed herein which encodes a tethered IL- 12 polypeptide comprising an IL- 12 polypeptide as disclosed herein and a membrane domain as disclosed herein.
  • the composition or formulation can contain a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a nucleic acid sequence (e.g., an ORF) having significant sequence identity to a sequence optimized nucleic acid sequence disclosed herein which encodes an IL- 12 polypeptide and/or a sequence optimized nucleic acid sequence disclosed herein which encodes a membrane domain.
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds miR-122.
  • compositions or formulation can optionally comprise one or more additional active substances, e.g. , therapeutically and/or prophylactically active substances.
  • Pharmaceutical compositions or formulation of the present disclosure can be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents can be found, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
  • compositions are administered to humans, human patients or subjects.
  • the phrase "active ingredient" generally refers to polynucleotides to be delivered as described herein.
  • Formulations and pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology.
  • such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition or formulation in accordance with the present disclosure can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a "unit dose" refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • the compositions and formulations described herein can contain at least one polynucleotide of the disclosure.
  • the composition or formulation can contain 1, 2, 3, 4 or 5 polynucleotides of the disclosure.
  • the compositions or formulations described herein can comprise more than one type of polynucleotide.
  • the composition or formulation can comprise a polynucleotide in linear and circular form.
  • the composition or formulation can comprise a circular polynucleotide and an IVT polynucleotide.
  • composition or formulation can comprise an IVT polynucleotide, a chimeric polynucleotide and a circular polynucleotide.
  • the present disclosure provides pharmaceutical formulations that comprise a polynucleotide described herein.
  • the polynucleotides described herein can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the polynucleotide); (4) alter the biodistribution (e.g., target the polynucleotide to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo.
  • the pharmaceutical formulation further comprises a delivery agent, (e.g., a compound having the Formula (I)).
  • a pharmaceutically acceptable excipient includes, but are not limited to, any and all solvents, dispersion media, or other liquid vehicles, dispersion or suspension aids, diluents, granulating and/or dispersing agents, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, binders, lubricants or oil, coloring, sweetening or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmolality adjusting agents, pH adjusting agents, buffers, chelants, cyoprotectants, and/or bulking agents, as suited to the particular dosage form desired.
  • Exemplary diluents include, but are not limited to, calcium or sodium carbonate, calcium phosphate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, etc., and/or combinations thereof.
  • Exemplary granulating and/or dispersing agents include, but are not limited to, starches, pregelatinized starches, or microcrystalline starch, alginic acid, guar gum, agar, polyvinylpyrrolidone), (providone), cross-linked poly(vinyl-pyrrolidone) (crospovidone), cellulose, methylcellulose, carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, etc., and/or combinations thereof.
  • Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], glyceryl monooleate, polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRU®30]), PLUORINC®F 68, POLOXAMER®188, etc. and/or combinations thereof.
  • natural emulsifiers e.g., a
  • Exemplary binding agents include, but are not limited to, starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), amino acids (e.g., glycine), natural and synthetic gums (e.g., acacia, sodium alginate), ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, etc., and combinations thereof.
  • sugars e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol
  • amino acids e.g., glycine
  • natural and synthetic gums e.g., acacia, sodium alginate
  • ethylcellulose hydroxyethylcellulose, hydroxypropyl methylcellulose, etc., and combinations thereof.
  • Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations.
  • antioxidants can be added to the formulations.
  • Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium or potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, etc., and combinations thereof.
  • Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, trisodium edetate, etc., and combinations thereof.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid monohydrate disodium edetate
  • fumaric acid malic acid
  • phosphoric acid sodium edetate
  • tartaric acid trisodium edetate, etc.
  • antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzethonium chloride, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, etc., and combinations thereof.
  • Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, butylated hydroxyanisol, ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), etc., and combinations thereof.
  • the pH of polynucleotide solutions are maintained between pH 5 and pH 8 to improve stability.
  • exemplary buffers to control pH can include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, etc., and/or combinations thereof.
  • Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium or magnesium lauryl sulfate, etc., and combinations thereof.

Abstract

La présente invention concerne des polynucléotides codant pour des polypeptides d'interleukine-12 (IL-12) ancrés comprenant un polypeptide d'IL-12 et un domaine membranaire. La présente invention concerne également des vecteurs comprenant les polynucléotides; des cellules hôtes comprenant les polynucléotides ou les vecteurs, des polypeptides codés par les polynucléotides; des compositions comprenant les polynucléotides, les vecteurs, les cellules hôtes, ou les polypeptides et un agent de distribution; et leurs utilisations, comprenant le traitement du cancer.
PCT/US2018/033436 2017-05-18 2018-05-18 Polynucléotides codant pour des polypeptides d'interleukine-12 (il12) ancrés et leurs utilisations WO2018213731A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP2019563454A JP7285220B2 (ja) 2017-05-18 2018-05-18 連結したインターロイキン－１２（ｉｌ１２）ポリペプチドをコードするポリヌクレオチドを含む脂質ナノ粒子
US16/613,732 US11421011B2 (en) 2017-05-18 2018-05-18 Polynucleotides encoding tethered interleukin-12 (IL12) polypeptides and uses thereof
CA3063723A CA3063723A1 (fr) 2017-05-18 2018-05-18 Polynucleotides codant pour des polypeptides d'interleukine-12 (il12) ancres et leurs utilisations
EP18729295.8A EP3625246A1 (fr) 2017-05-18 2018-05-18 Polynucléotides codant pour des polypeptides d'interleukine-12 (il12) ancrés et leurs utilisations
AU2018270111A AU2018270111B2 (en) 2017-05-18 2018-05-18 Polynucleotides encoding tethered interleukin-12 (IL12) polypeptides and uses thereof
US17/875,887 US11873327B2 (en) 2017-05-18 2022-07-28 Polynucleotides encoding tethered interleukin-12 (IL12) polypeptides and uses thereof
JP2023015202A JP2023059880A (ja) 2017-05-18 2023-02-03 連結したインターロイキン－１２（ｉｌ１２）ポリペプチドをコードするポリヌクレオチド及びその使用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762508316P 2017-05-18 2017-05-18
US62/508,316 2017-05-18

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US16/613,732 A-371-Of-International US11421011B2 (en) 2017-05-18 2018-05-18 Polynucleotides encoding tethered interleukin-12 (IL12) polypeptides and uses thereof
US17/875,887 Division US11873327B2 (en) 2017-05-18 2022-07-28 Polynucleotides encoding tethered interleukin-12 (IL12) polypeptides and uses thereof

Publications (1)

Publication Number Publication Date
WO2018213731A1 true WO2018213731A1 (fr) 2018-11-22

Family

ID=62528895

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/033436 WO2018213731A1 (fr) 2017-05-18 2018-05-18 Polynucléotides codant pour des polypeptides d'interleukine-12 (il12) ancrés et leurs utilisations

Country Status (6)

Country Link
US (2) US11421011B2 (fr)
EP (1) EP3625246A1 (fr)
JP (2) JP7285220B2 (fr)
AU (1) AU2018270111B2 (fr)
CA (1) CA3063723A1 (fr)
WO (1) WO2018213731A1 (fr)

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110095463A (zh) * 2019-03-15 2019-08-06 中国人民解放军陆军军医大学第二附属医院 一种乳糜定性检测的试剂盒
EP3523323A4 (fr) * 2016-10-07 2020-06-17 Board of Regents, The University of Texas System Lymphocytes t exprimant il-12 ancrée sur membrane pour le traitement du cancer
WO2020123716A1 (fr) 2018-12-11 2020-06-18 Obsidian Therapeutics, Inc. Il12 liée à la membrane, compositions et procédés de régulation accordable
WO2020160350A1 (fr) * 2019-02-01 2020-08-06 Board Of Regents, The University Of Texas System Thérapie par lymphocytes t à il-12 modifiés pour le traitement du cancer
WO2020252404A1 (fr) 2019-06-12 2020-12-17 Obsidian Therapeutics, Inc. Compositions de ca2 et procédés de régulation accordable
WO2021072172A1 (fr) * 2019-10-09 2021-04-15 Translate Bio, Inc. Compositions, méthodes et utilisations d'arn messager
US10993967B2 (en) 2018-10-17 2021-05-04 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
US11058725B2 (en) 2019-09-10 2021-07-13 Obsidian Therapeutics, Inc. CA2 compositions and methods for tunable regulation
WO2021221127A1 (fr) * 2020-04-30 2021-11-04 Vlp Therapeutics, Inc. Immunothérapie à cytokine
WO2021262985A1 (fr) * 2020-06-25 2021-12-30 Amunix Pharmaceuticals, Inc. Conjugués de cytokine
WO2022020811A1 (fr) * 2020-07-24 2022-01-27 Strand Therapeutics, Inc. Nanoparticule de nanoparticule lipidique comprenant des nucléotides modifiés
US11358999B2 (en) 2018-10-03 2022-06-14 Xencor, Inc. IL-12 heterodimeric Fc-fusion proteins
US11419898B2 (en) 2018-10-17 2022-08-23 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
US11446332B2 (en) 2017-04-13 2022-09-20 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
US11661459B2 (en) 2020-12-03 2023-05-30 Century Therapeutics, Inc. Artificial cell death polypeptide for chimeric antigen receptor and uses thereof
WO2023196988A1 (fr) * 2022-04-07 2023-10-12 Modernatx, Inc. Procédés d'utilisation d'arnm codant pour il-12
US11851466B2 (en) 2019-10-03 2023-12-26 Xencor, Inc. Targeted IL-12 heterodimeric Fc-fusion proteins
US11883432B2 (en) 2020-12-18 2024-01-30 Century Therapeutics, Inc. Chimeric antigen receptor system with adaptable receptor specificity

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3063723A1 (fr) 2017-05-18 2018-11-22 Modernatx, Inc. Polynucleotides codant pour des polypeptides d'interleukine-12 (il12) ancres et leurs utilisations
CA3068841A1 (fr) 2017-07-03 2019-01-10 Torque Therapeutics, Inc. Molecules de fusion ciblant des cellules regulatrices immunitaires et leurs utilisations
AU2019338535A1 (en) * 2018-09-14 2021-04-15 Modernatx, Inc. Methods and compositions for treating cancer using mRNA therapeutics
JP2023548586A (ja) * 2020-11-04 2023-11-17 センティ バイオサイエンシズ インコーポレイテッド タンパク質ペイロード放出
WO2023205614A2 (fr) * 2022-04-21 2023-10-26 Senti Biosciences, Inc. Systèmes d'expression de protéines chimériques multicistroniques
WO2023229013A1 (fr) * 2022-05-27 2023-11-30 北京天一方生物科技▲発▼展有限公司 Procédé de production de cellules myéloïdes prolifératives qui produisent de manière constitutive il-12p70
WO2024059629A1 (fr) * 2022-09-13 2024-03-21 Imunon, Inc. Combinaison de thérapie génique utilisant l'il-12 et d'inhibiteurs de point de contrôle immunitaire pour le traitement du cancer

Citations (205)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5082767A (en) 1989-02-27 1992-01-21 Hatfield G Wesley Codon pair utilization
US5505931A (en) 1993-03-04 1996-04-09 The Dow Chemical Company Acid cleavable compounds, their preparation and use as bifunctional acid-labile crosslinking agents
US5563250A (en) 1987-12-02 1996-10-08 Neorx Corporation Cleavable conjugates for the delivery and release of agents in native form
US5585108A (en) 1994-12-30 1996-12-17 Nanosystems L.L.C. Formulations of oral gastrointestinal therapeutic agents in combination with pharmaceutically acceptable clays
US5681702A (en) 1994-08-30 1997-10-28 Chiron Corporation Reduction of nonspecific hybridization by using novel base-pairing schemes
US6004573A (en) 1997-10-03 1999-12-21 Macromed, Inc. Biodegradable low molecular weight triblock poly(lactide-co-glycolide) polyethylene glycol copolymers having reverse thermal gelation properties
WO2000018778A1 (fr) 1998-09-29 2000-04-06 Phylos, Inc. Synthese d'acides nucleiques fondee sur une generation aleatoire de codons
US6177274B1 (en) 1998-05-20 2001-01-23 Expression Genetics, Inc. Hepatocyte targeting polyethylene glyco-grafted poly-L-lysine polymeric gene carrier
US6217912B1 (en) 1998-07-13 2001-04-17 Expression Genetics, Inc. Polyester analogue of poly-L-lysine as a soluble, biodegradable gene delivery carrier
US6265389B1 (en) 1995-08-31 2001-07-24 Alkermes Controlled Therapeutics, Inc. Microencapsulation and sustained release of oligonucleotides
US6267987B1 (en) 1997-12-12 2001-07-31 Samyang Corporation Positively charged poly[alpha-(omega-aminoalkyl) glycolic acid] for the delivery of a bioactive agent via tissue and cellular uptake
US6426086B1 (en) 1998-02-03 2002-07-30 The Regents Of The University Of California pH-sensitive, serum-stable liposomes
US6517869B1 (en) 1997-12-12 2003-02-11 Expression Genetics, Inc. Positively charged poly(alpha-(omega-aminoalkyl)lycolic acid) for the delivery of a bioactive agent via tissue and cellular uptake
US20030073619A1 (en) 2000-09-14 2003-04-17 Mahato Ram I. Novel cationic lipopolymer as biocompatible gene delivery agent
US6586524B2 (en) 2001-07-19 2003-07-01 Expression Genetics, Inc. Cellular targeting poly(ethylene glycol)-grafted polymeric gene carrier
US6652886B2 (en) 2001-02-16 2003-11-25 Expression Genetics Biodegradable cationic copolymers of poly (alkylenimine) and poly (ethylene glycol) for the delivery of bioactive agents
US20040142474A1 (en) 2000-09-14 2004-07-22 Expression Genetics, Inc. Novel cationic lipopolymer as a biocompatible gene delivery agent
US6835393B2 (en) 1998-01-05 2004-12-28 University Of Washington Enhanced transport using membrane disruptive agents
US20040262223A1 (en) 2001-07-27 2004-12-30 President And Fellows Of Harvard College Laminar mixing apparatus and methods
US20050032730A1 (en) 2001-06-05 2005-02-10 Florian Von Der Mulbe Pharmaceutical composition containing a stabilised mRNA optimised for translation in its coding regions
US20050059005A1 (en) 2001-09-28 2005-03-17 Thomas Tuschl Microrna molecules
US6897196B1 (en) 2001-02-07 2005-05-24 The Regents Of The University Of California pH sensitive lipids based on ortho ester linkers, composition and method
US20050222064A1 (en) 2002-02-20 2005-10-06 Sirna Therapeutics, Inc. Polycationic compositions for cellular delivery of polynucleotides
US20050261218A1 (en) 2003-07-31 2005-11-24 Christine Esau Oligomeric compounds and compositions for use in modulation small non-coding RNAs
US6984720B1 (en) 1999-08-24 2006-01-10 Medarex, Inc. Human CTLA-4 antibodies
WO2006063249A2 (fr) 2004-12-10 2006-06-15 Justin Hanes Copolymeres sequences de poly(ether-anhydride) fonctionnalises
US7098032B2 (en) 2001-01-02 2006-08-29 Mirus Bio Corporation Compositions and methods for drug delivery using pH sensitive molecules
US7138382B2 (en) 1999-06-07 2006-11-21 Mirus Bio Corporation Compositions and methods for drug delivery using pH sensitive molecules
WO2007024323A2 (fr) 2005-06-17 2007-03-01 The University Of North Carolina At Chapel Hill Procedes, systemes et materiaux de fabrication de nanoparticules
US20070117112A1 (en) 2005-06-30 2007-05-24 Diener John L Materials and methods for the generation of fully 2'-modified nucleic acid transcripts
WO2007113648A2 (fr) 2006-04-05 2007-10-11 Pfizer Products Inc. Polythérapie à base d'un anticorps anti-ctla4
US20080046192A1 (en) 2006-08-16 2008-02-21 Richard Lathrop Polypepetide-encoding nucleotide sequences with refined translational kinetics and methods of making same
US20080076161A1 (en) 2002-04-01 2008-03-27 The Government Of The United States, As Represented By The Secretary Of The Army Method of designing synthetic nucleic acid sequences for optimal protein expression in a host cell
WO2008078180A2 (fr) 2006-12-22 2008-07-03 Archemix Corp. Matériaux et procédés pour la génération de transcrits comprenant des nucléotides modifiés
US7404969B2 (en) 2005-02-14 2008-07-29 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
WO2008103276A2 (fr) 2007-02-16 2008-08-28 Merck & Co., Inc. Compositions et méthodes de potentialisation de l'activité de molécules biologiquement actives
WO2008121949A1 (fr) 2007-03-30 2008-10-09 Bind Biosciences, Inc. Ciblage de cellules cancéreuses utilisant des nanoparticules
US7561973B1 (en) 2008-07-31 2009-07-14 Dna Twopointo, Inc. Methods for determining properties that affect an expression property value of polynucleotides in an expression system
US20090226470A1 (en) 2007-12-11 2009-09-10 Mauro Vincent P Compositions and methods related to mRNA translational enhancer elements
US20100004315A1 (en) 2008-03-14 2010-01-07 Gregory Slobodkin Biodegradable Cross-Linked Branched Poly(Alkylene Imines)
WO2010005723A2 (fr) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Nanoparticules polymères pharmacologiquement chargées et leurs méthodes de fabrication et d’utilisation
US20100009424A1 (en) 2008-07-14 2010-01-14 Natasha Forde Sonoporation systems and methods
WO2010005725A2 (fr) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Nanoparticules polymères thérapeutiques comprenant des alcaloïdes vinca et procédés de fabrication et d’utilisation associés
WO2010005740A2 (fr) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Procédés pour la préparation de copolymères diblocs fonctionnalisés avec un agent de ciblage destinés à être utilisés dans la fabrication de nanoparticules ciblées thérapeutiques
WO2010005726A2 (fr) 2008-06-16 2010-01-14 Bind Biosciences Inc. Nanoparticules polymères thérapeutiques avec inhibiteurs de mtor et procédés de fabrication et d’utilisation associés
US20100036115A1 (en) 1997-07-23 2010-02-11 Sirna Therapeutics, Inc. Novel Compositions for the Delivery of Negatively Charged Molecules
WO2010021865A1 (fr) 2008-08-18 2010-02-25 Merck Sharp & Dohme Corp. Nouvelles nanoparticules lipidiques et nouveaux composants pour l'administration d'acides nucléiques
WO2010030763A2 (fr) 2008-09-10 2010-03-18 Bind Biosciences, Inc. Fabrication de nanoparticles à rendement élevé
CN101684456A (zh) * 2008-09-28 2010-03-31 江门罗森生物制药有限公司 一种体外培养条件下扩增人nk细胞的方法
WO2010047839A1 (fr) 2008-10-25 2010-04-29 Aura Biosciences Particules de virus de plante modifiées et utilisations de celles-ci
US20100120024A1 (en) 2005-06-30 2010-05-13 Sharon Cload Materials and methods for the generation of transcripts comprising modified nucleotides
US20100129877A1 (en) 2005-09-28 2010-05-27 Ugur Sahin Modification of RNA, Producing an Increased Transcript Stability and Translation Efficiency
US7737108B1 (en) 2000-01-07 2010-06-15 University Of Washington Enhanced transport using membrane disruptive agents
WO2010075072A2 (fr) 2008-12-15 2010-07-01 Bind Biosciences Longues nanoparticules circulantes pour la libération prolongée d'agents thérapeutiques
WO2010080724A1 (fr) 2009-01-12 2010-07-15 Merck Sharp & Dohme Corp. Nanoparticules lipidiques inédites et composants inédits utilisables pour l'administration d'acides nucléiques
WO2010087791A1 (fr) 2009-01-27 2010-08-05 Utc Power Corporation Réacteur de conversion à la vapeur d'eau intégré, refroidi de manière distributive et atomiseur
US20100196983A1 (en) 2009-02-05 2010-08-05 Ut-Battelle, Llc Transformation of gram positive bacteria by sonoporation
WO2010120266A1 (fr) 2009-04-13 2010-10-21 Inserm, Institut National De La Sante Et De La Recherche Medicale Particules hpv et utilisations associées
US20100293625A1 (en) 2007-09-26 2010-11-18 Interexon Corporation Synthetic 5'UTRs, Expression Vectors, and Methods for Increasing Transgene Expression
US20100324120A1 (en) 2009-06-10 2010-12-23 Jianxin Chen Lipid formulation
US7893302B2 (en) 2005-02-14 2011-02-22 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
WO2011022460A1 (fr) 2009-08-20 2011-02-24 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques avec différents groupes de tête pour délivrance d’oligonucléotide
WO2011043913A2 (fr) 2009-10-08 2011-04-14 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques à chaînes lipidiques courtes pour une administration d'oligonucléotides
US7943743B2 (en) 2005-07-01 2011-05-17 Medarex, Inc. Human monoclonal antibodies to programmed death ligand 1 (PD-L1)
WO2011062965A2 (fr) 2009-11-18 2011-05-26 University Of Washington Through Its Center For Commercialization Monomères de ciblage et polymère ayant des blocs de ciblage
WO2011084518A2 (fr) 2009-12-15 2011-07-14 Bind Biosciences, Inc. Nanoparticules polymères thérapeutiques comprenant de corticostéroïdes, et procédés pour les fabriquer et les utiliser
US20110171248A1 (en) 2010-01-08 2011-07-14 Selecta Biosciences, Inc. Synthetic virus-like particles conjugated to human papillomavirus capsid peptides for use as vaccines
WO2011084521A2 (fr) 2009-12-15 2011-07-14 Bind Biosciences, Inc. Nanoparticules polymères thérapeutiques comportant de l'épothilone et leurs procédés de fabrication et d'utilisation
WO2011090965A1 (fr) 2010-01-22 2011-07-28 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques pour transfert d'oligonucléotide
US8008449B2 (en) 2005-05-09 2011-08-30 Medarex, Inc. Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics
WO2011127255A1 (fr) 2010-04-08 2011-10-13 Merck Sharp & Dohme Corp. Préparation de nanoparticules de lipide
US20110262491A1 (en) 2010-04-12 2011-10-27 Selecta Biosciences, Inc. Emulsions and methods of making nanocarriers
US8057821B2 (en) 2004-11-03 2011-11-15 Egen, Inc. Biodegradable cross-linked cationic multi-block copolymers for gene delivery and methods of making thereof
WO2011149733A2 (fr) 2010-05-24 2011-12-01 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques alcools aminés pour l'administration d'oligonucléotides
WO2011153120A1 (fr) 2010-06-04 2011-12-08 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques de faible poids moléculaire pour l'administration d'oligonucléotides
WO2012006380A2 (fr) 2010-07-06 2012-01-12 Novartis Ag Émulsions cationiques huile-dans-eau
WO2012006378A1 (fr) 2010-07-06 2012-01-12 Novartis Ag Liposomes à lipides ayant une valeur de pka avantageuse pour la délivrance d'arn
WO2012006376A2 (fr) 2010-07-06 2012-01-12 Novartis Ag Particules d'administration de type virion pour des molécules d'arn auto-répliquant
WO2012013326A1 (fr) 2010-07-30 2012-02-02 Curevac Gmbh Complexation d'acides nucléiques avec des composants cationiques réticulés par un pont disulfure pour une transfection et une immunostimulation
US8126653B2 (en) 2008-07-31 2012-02-28 Dna Twopointo, Inc. Synthetic nucleic acids for expression of encoded proteins
WO2012031043A1 (fr) 2010-08-31 2012-03-08 Novartis Ag Liposomes pégylés pour l'apport d'arn codant pour un immunogène
WO2012031046A2 (fr) 2010-08-31 2012-03-08 Novartis Ag Lipides adaptés pour une administration liposomale d'arn codant pour une protéine
WO2012030901A1 (fr) 2010-08-31 2012-03-08 Novartis Ag Petits liposomes destinés à l'administration d'un arn codant pour un immunogène
WO2012040184A2 (fr) 2010-09-20 2012-03-29 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques de faible poids moléculaire pour l'administration d'oligonucléotides
WO2012040524A1 (fr) 2010-09-24 2012-03-29 Mallinckrodt Llc Conjugués d'aptamères pour ciblage de nanovecteurs thérapeutiques et/ou diagnostiques
WO2012044638A1 (fr) 2010-09-30 2012-04-05 Merck Sharp & Dohme Corp. Lipides cationiques de faible masse moléculaire utilisables en vue de l'administration d'oligonucléotides
WO2012049366A1 (fr) 2010-10-14 2012-04-19 Timo Vesikari Protéine de capside de norovirus et de rotavirus vp6 a usage comme vaccin combine
WO2012054923A2 (fr) 2010-10-22 2012-04-26 Bind Biosciences, Inc. Nanoparticules thérapeutiques contenant des copolymères de masse moléculaire élevée
WO2012054365A2 (fr) 2010-10-21 2012-04-26 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques à faible poids moléculaire destinés à une administration d'oligonucléotides
WO2012061259A2 (fr) 2010-11-05 2012-05-10 Merck Sharp & Dohme Corp. Amine cyclique inédite de faible masse moléculaire contenant des lipides cationiques en vue de l'administration d'oligonucléotides
US20120140790A1 (en) 2009-12-15 2012-06-07 Ali Mir M Therapeutic Polymeric Nanoparticle Compositions with High Glass Transition Termperature or High Molecular Weight Copolymers
US8206749B1 (en) 1999-02-26 2012-06-26 Novartis Vaccines And Diagnostics, Inc. Microemulsions with adsorbed macromolecules and microparticles
US8205511B1 (en) 2003-10-17 2012-06-26 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Air-sampling device and method of use
US8217149B2 (en) 2008-12-09 2012-07-10 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
US20120178702A1 (en) 1995-01-23 2012-07-12 University Of Pittsburgh Stable lipid-comprising drug delivery complexes and methods for their production
US8236280B2 (en) 2003-12-19 2012-08-07 University Of Cincinnati Polyamides for nucleic acid delivery
US20120202871A1 (en) 2009-07-01 2012-08-09 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US20120201859A1 (en) 2002-05-02 2012-08-09 Carrasquillo Karen G Drug Delivery Systems and Use Thereof
US8241670B2 (en) 2004-04-15 2012-08-14 Chiasma Inc. Compositions capable of facilitating penetration across a biological barrier
WO2012109121A1 (fr) 2011-02-07 2012-08-16 Purdue Research Foundation Nanoparticules glucidiques pour une efficacité prolongée d'un peptide antimicrobien
US20120207840A1 (en) 2011-02-10 2012-08-16 Aura Biosciences, Inc. Virion Derived Protein Nanoparticles For Delivering Diagnostic Or Therapeutic Agents For The Treatment Of Non-Melanoma Skin Cancer
WO2012110636A2 (fr) 2011-02-18 2012-08-23 Instituto Nacional De Investigación Y Tecnología Agraria Y Alimentaria (Inia) Peptides supports pour distribution dans cellules
US8257745B2 (en) 2001-12-21 2012-09-04 Novartis Ag Use of synthetic inorganic nanoparticles as carriers for ophthalmic and otic drugs
WO2012122444A1 (fr) 2011-03-10 2012-09-13 Provectus Pharmaceuticals, Inc. Combinaison de thérapies immunomodulatrices locales et systémiques pour l'amélioration du traitement du cancer
US20120228565A1 (en) 2000-10-13 2012-09-13 Life Technologies Corporation Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
WO2012135805A2 (fr) 2011-03-31 2012-10-04 modeRNA Therapeutics Administration et formulation d'acides nucléiques génétiquement modifiés
US8283333B2 (en) 2009-07-01 2012-10-09 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
US20120258046A1 (en) 2009-12-09 2012-10-11 Thorsten Mutzke Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids
US20120265001A1 (en) 2010-10-11 2012-10-18 Wichita State University Composite magnetic nanoparticle drug delivery system
US20120276209A1 (en) 2009-11-04 2012-11-01 The University Of British Columbia Nucleic acid-containing lipid particles and related methods
US20120283503A1 (en) 2011-04-29 2012-11-08 The Johns Hopkins University Nanoparticle loaded stem cells and their use in mri guided hyperthermia
US8313777B2 (en) 2006-10-05 2012-11-20 The Johns Hopkins University Water-dispersible oral, parenteral, and topical formulations for poorly water soluble drugs using smart polymeric nanoparticles
US20120295832A1 (en) 2011-05-17 2012-11-22 Arrowhead Research Corporation Novel Lipids and Compositions for Intracellular Delivery of Biologically Active Compounds
WO2012170930A1 (fr) 2011-06-08 2012-12-13 Shire Human Genetic Therapies, Inc Compositions de nanoparticules lipides et procédés pour le transfert d'arnm
US20130012450A1 (en) 2011-07-10 2013-01-10 Aura Biosciences, Inc. Virion Derived Protein Nanoparticles For Delivering Diagnostic Or Therapeutic Agents For The Treatment Of Dermatology Related Genetic Diseases
WO2013009717A1 (fr) 2011-07-10 2013-01-17 Elisabet De Los Pinos Nanoparticules de protéine virionique pour l'administration d'agents diagnostiques ou thérapeutiques pour le traitement de maladies cutanées
US20130017199A1 (en) 2009-11-24 2013-01-17 AMPLIMMUNE ,Inc. a corporation Simultaneous inhibition of pd-l1/pd-l2
WO2013007604A1 (fr) 2011-07-08 2013-01-17 Bayer Intellectual Property Gmbh Procédé de production de dérivés de diamide d'acide anthranilique substitués par tétrazole par mise en réaction d'acides pyrazoliques avec des esters d'acide anthranilique
US20130059360A1 (en) 2005-04-12 2013-03-07 Nektar Therapeutics Polymer-based compositions and conjugates of antimicrobial agents
WO2013033438A2 (fr) 2011-08-31 2013-03-07 Mallinckrodt Llc Modification de nanoparticules de peg avec des h-phosphonates
US20130064894A1 (en) 2011-08-31 2013-03-14 Protiva Biotherapeutics, Inc. Novel cationic lipids and methods of use thereof
US8401798B2 (en) 2008-06-06 2013-03-19 Dna Twopointo, Inc. Systems and methods for constructing frequency lookup tables for expression systems
WO2013039857A1 (fr) 2011-09-12 2013-03-21 modeRNA Therapeutics Acides nucléiques modifiés et leurs procédés d'utilisation
US20130072709A1 (en) 2006-02-21 2013-03-21 Nektar Therapeutics Segmented Degradable Polymers and Conjugates Made Therefrom
US8404222B2 (en) 1996-09-26 2013-03-26 Nektar Therapeutics Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution
WO2013052523A1 (fr) 2011-10-03 2013-04-11 modeRNA Therapeutics Nucléosides, nucléotides et acides nucléiques modifiés, et leurs utilisations
US20130116408A1 (en) 2011-11-05 2013-05-09 Aura Biosciences, Inc. Virion Derived Protein Nanoparticles For Delivering Radioisotopes For The Diagnosis And Treatment Of Malignant And Systemic Disease And The Monitoring Of Therapy
US20130115247A1 (en) 2011-11-05 2013-05-09 Aura Biosciences, Inc. Virion Derived Protein Nanoparticles For Delivering Radioisotopes For The Diagnosis And Treatment Of Malignant And Systemic Disease And The Monitoring Of Therapy
US20130123338A1 (en) 2010-05-12 2013-05-16 Protiva Biotherapeutics, Inc. Novel cationic lipids and methods of use thereof
US8444992B2 (en) 2005-09-01 2013-05-21 Novartis Vaccines And Diagnostics Gmbh Multiple vaccination including serogroup C meningococcus
WO2013075068A1 (fr) 2011-11-18 2013-05-23 Regeneron Pharmaceuticals, Inc. Microparticules de protéine et de polymère
US20130130348A1 (en) 2006-05-15 2013-05-23 The Brigham And Women's Hospital, Inc. Polymers for Functional Particles
US20130129726A1 (en) 2006-02-20 2013-05-23 Kyunglim Lee Peptide having cell membrane penetrating activity
US20130129785A1 (en) 2010-05-10 2013-05-23 Alnylam Pharmaceuticals, Inc Methods and compositions for delivery of active agents
US8450298B2 (en) 2008-11-07 2013-05-28 Massachusetts Institute Of Technology Aminoalcohol lipidoids and uses thereof
US20130137644A1 (en) 2005-12-16 2013-05-30 Cellectis Cell penetrating peptide conjugates for delivering of nucleic acids into a cell
WO2013082111A2 (fr) 2011-11-29 2013-06-06 The University Of North Carolina At Chapel Hill Particules manipulées de façon géométrique et procédés de modulation de réponses des macrophages ou immunitaires
US8460696B2 (en) 2009-03-20 2013-06-11 Egen, Inc. Polyamine derivatives
US8460709B2 (en) 2002-03-13 2013-06-11 Novartis Ag Pharmaceutical microparticles
US20130150295A1 (en) 2006-12-21 2013-06-13 Stryker Corporation Sustained-Release Formulations Comprising Crystals, Macromolecular Gels, and Particulate Suspensions of Biologic Agents
WO2013086322A1 (fr) 2011-12-07 2013-06-13 Alnylam Pharmaceuticals, Inc. Lipides biodégradables ramifiés à terminaisons alkyle et cycloalkyle destinés à l'administration d'agents actifs
WO2013084000A2 (fr) 2011-12-07 2013-06-13 Isis Innovation Limited Exosomes pour l'administration de biothérapiques
WO2013086373A1 (fr) 2011-12-07 2013-06-13 Alnylam Pharmaceuticals, Inc. Lipides pour l'administration d'agents actifs
WO2013086526A1 (fr) 2011-12-09 2013-06-13 The Regents Of The University Of California Encapsulation liposomale de médicaments
US8466122B2 (en) 2010-09-17 2013-06-18 Protiva Biotherapeutics, Inc. Trialkyl cationic lipids and methods of use thereof
WO2013090648A1 (fr) 2011-12-16 2013-06-20 modeRNA Therapeutics Nucléoside, nucléotide, et compositions d'acide nucléique modifiés
WO2013088250A1 (fr) 2011-12-13 2013-06-20 Engeneic Molecular Delivery Pty Ltd Minicellules intactes, d'origine bactérienne, pour l'administration d'agents thérapeutiques à des tumeurs cérébrales
US20130164219A1 (en) 2010-06-14 2013-06-27 Hoffmann-La Roche Inc. Cell-penetrating peptides and uses thereof
US20130171646A1 (en) 2010-08-09 2013-07-04 So Jung PARK Nanop article-oligonucleotide hybrid structures and methods of use thereof
US20130177635A1 (en) 2010-04-09 2013-07-11 Pacira Pharmaceuticals, Inc. Method for formulating large diameter synthetic membrane vesicles
US20130177611A1 (en) 2004-06-11 2013-07-11 Eidgenossisches Technische Hochschule (The Swiss Federal Institute of Technology) Silk-based drug delivery system
US20130177587A1 (en) 2003-07-11 2013-07-11 Novavax, Inc. Functional influenza virus-like particles (vlps)
WO2013106525A1 (fr) 2012-01-12 2013-07-18 Stc.Unm Vlp immunogènes contenant le hpv l2 et compositions et procédés associés
WO2013106072A1 (fr) 2012-01-10 2013-07-18 Sorbent Therapeutics, Inc. Compositions comprenant des polymères réticulés de liaison à des cations et utilisations associées
WO2013106086A1 (fr) 2012-01-10 2013-07-18 Sorbent Therapeutics, Inc. Compositions comprenant des polymères réticulés de liaison à des cations et utilisations associées
US20130183244A1 (en) 2010-09-10 2013-07-18 The Johns Hopkins University Rapid Diffusion of Large Polymeric Nanoparticles in the Mammalian Brain
WO2013106073A1 (fr) 2012-01-10 2013-07-18 Sorbent Therapeutics, Inc. Compositions comprenant des polymères réticulés de liaison à des cations et utilisations associées
US20130184453A1 (en) 1998-07-01 2013-07-18 California Institute Of Technology Linear cyclodextrin copolymers
WO2013105101A1 (fr) 2012-01-13 2013-07-18 Department Of Biotechnology Nanoparticules lipidiques solides encapsulant un médicament hydrophile/amphiphile et leur procédé de préparation
WO2013110028A1 (fr) 2012-01-19 2013-07-25 The Johns Hopkins University Formulations de nanoparticules présentant une pénétration améliorée dans les muqueuses
US8496945B2 (en) 2007-03-05 2013-07-30 Washington University Nanoparticle delivery systems for membrane-integrating peptides
US20130196948A1 (en) 2010-06-25 2013-08-01 Massachusetts Insitute Of Technology Polymers for biomaterials and therapeutics
US20130195799A1 (en) 2010-08-19 2013-08-01 Peg Biosciences, Inc. Synergistic biomolecule-polymer conjugates
US20130195920A1 (en) 2011-12-07 2013-08-01 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US8501824B2 (en) 2007-05-04 2013-08-06 Marina Biotech, Inc. Amino acid lipids and uses thereof
WO2013116656A1 (fr) 2012-02-03 2013-08-08 Emory University Compositions immunostimulatrices, particules et applications associées
US8506967B2 (en) 2003-07-11 2013-08-13 Novavax, Inc. Functional influenza virus like particles (VLPs)
US8507653B2 (en) 2006-12-27 2013-08-13 Nektar Therapeutics Factor IX moiety-polymer conjugates having a releasable linkage
WO2013119877A1 (fr) 2012-02-07 2013-08-15 Aura Biosciences, Inc. Nanosphères dérivées de virions pour l'administration sélective d'agents thérapeutiques et diagnostiques dans des cellules cancéreuses
US20130211249A1 (en) 2010-07-22 2013-08-15 The Johns Hopkins University Drug eluting hydrogels for catheter delivery
WO2013119602A1 (fr) 2012-02-06 2013-08-15 President And Fellows Of Harvard College Microvésicules médiées par arrdc1 (armm) et leurs utilisations
US20130209544A1 (en) 2010-05-26 2013-08-15 Micromedmark Biotech Co., Ltd. Microvesicles carrying small interfering rnas, preparation methods and uses thereof
WO2013123298A1 (fr) 2012-02-17 2013-08-22 University Of Georgia Research Foundation, Inc. Nanoparticules de trafic mitochondrial d'agents
WO2013123523A1 (fr) 2012-02-19 2013-08-22 Nvigen, Inc. Utilisations d'une nanostructure poreuse lors de son administration
US20130217753A1 (en) 2011-02-22 2013-08-22 Rutgers, The State University Of New Jersey Amphiphilic macromolecules for nucleic acid delivery
WO2013122262A1 (fr) 2012-02-16 2013-08-22 Vlp Therapeutics, Llc Composition à base d'une particule de type viral
US8519110B2 (en) 2008-06-06 2013-08-27 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College mRNA cap analogs
WO2013124867A1 (fr) 2012-02-21 2013-08-29 Amrita Vishwa Vidyapeetham University Polymer - polymer or polymer - protein core - shell nano medicine loaded with multiple drug molecules
WO2013126803A1 (fr) 2012-02-24 2013-08-29 Protiva Biotherapeutics Inc. Lipides cationiques trialkylés et leurs procédés d'utilisation
US8524368B2 (en) 2003-07-09 2013-09-03 Wisconsin Alumni Research Foundation Charge-dynamic polymers and delivery of anionic compounds
US8524259B2 (en) 2006-12-05 2013-09-03 Landec Corporation Systems and methods for delivery of materials
US20130231287A1 (en) 2010-02-25 2013-09-05 Parimala Nacharaju Pegylated albumin polymers and uses thereof
US20130259923A1 (en) 2012-04-02 2013-10-03 modeRNA Therapeutics Modified polynucleotides for the production of secreted proteins
WO2013151666A2 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés destinés à la production de produits biologiques et de protéines associées à une maladie humaine
WO2013173223A1 (fr) 2012-05-15 2013-11-21 Bristol-Myers Squibb Company Immunothérapie anticancéreuse par rupture de la signalisation pd-1/pd-l1
US8609089B2 (en) 2008-08-25 2013-12-17 Amplimmune, Inc. Compositions of PD-1 antagonists and methods of use
WO2014028429A2 (fr) 2012-08-14 2014-02-20 Moderna Therapeutics, Inc. Enzymes et polymérases destinées à la synthèse d'arn
US20140121263A1 (en) 2009-06-15 2014-05-01 Alnylam Pharmaceuticals, Inc. Lipid formulated dsrna targeting the pcsk9 gene
WO2014093924A1 (fr) 2012-12-13 2014-06-19 Moderna Therapeutics, Inc. Molécules d'acide nucléique modifiées et leurs utilisations
US20140200261A1 (en) 2013-01-17 2014-07-17 Moderna Therapeutics, Inc. Signal-sensor polynucleotides for the alteration of cellular phenotypes
US20140228558A1 (en) 2002-12-23 2014-08-14 Geneart Ag Method and device for optimizing a nucelotide sequence for the purpose of expression in a protein
WO2014152211A1 (fr) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation et administration de compositions de nucléosides, de nucléotides, et d'acides nucléiques modifiés
WO2014164253A1 (fr) 2013-03-09 2014-10-09 Moderna Therapeutics, Inc. Régions non traduites hétérologues pour arnm
US20140341917A1 (en) 2011-11-28 2014-11-20 Merck Patent Gmbh Anti-pd-l1 antibodies and uses thereof
US8900587B2 (en) 2007-06-18 2014-12-02 Merck Sharp & Dohme Corp. Antibodies to human programmed death receptor PD-1
US20140363493A1 (en) 2013-06-10 2014-12-11 Albert Einstein College Of Medicine Of Yeshiva University LIPID NANOPARTICLES FOR TARGETED siRNA DELIVERY
WO2015011633A1 (fr) 2013-07-23 2015-01-29 Protiva Biotherapeutics, Inc. Compositions et procédés pour l'administration d'arn messager
US20150079109A1 (en) 2013-09-13 2015-03-19 Beigene, Ltd. Anti-PD1 Antibodies and their Use as Therapeutics and Diagnostics
WO2015051214A1 (fr) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucléotides codant pour un récepteur de lipoprotéines de faible densité
WO2015123264A1 (fr) 2014-02-11 2015-08-20 Alnylam Pharmaceuticals, Inc. Compositions d'arni de cétohexokinase (khk) et procédés pour les utiliser
WO2015130584A2 (fr) 2014-02-25 2015-09-03 Merck Sharp & Dohme Corp. Adjuvants de vaccins sous forme de nanoparticules lipidiques et systèmes d'administration d'antigènes
WO2015148247A1 (fr) 2014-03-24 2015-10-01 Shire Human Genetic Therapies, Inc. Thérapie basée sur l'arnm pour le traitement des maladies oculaires
WO2015199952A1 (fr) 2014-06-25 2015-12-30 Acuitas Therapeutics Inc. Nouveaux lipides et formulations nanoparticulaires lipidiques pour l'administration d'acides nucléiques
WO2016048903A1 (fr) * 2014-09-22 2016-03-31 Intrexon Corporation Contrôle thérapeutique amélioré de formes hétérodimères et à chaîne unique de l'interleukine-12
WO2017004924A1 (fr) 2015-07-07 2017-01-12 卫小会 Procédé et système de communication pour le partage d'informations de navigation

Family Cites Families (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5891680A (en) 1995-02-08 1999-04-06 Whitehead Institute For Biomedical Research Bioactive fusion proteins comprising the p35 and p40 subunits of IL-12
US5922685A (en) 1996-06-05 1999-07-13 Powderject Vaccines, Inc. IL-12 gene therapy of tumors
AU4908197A (en) 1996-10-18 1998-05-15 Valentis, Inc. Gene expression and delivery systems and uses
US5994104A (en) 1996-11-08 1999-11-30 Royal Free Hospital School Of Medicine Interleukin-12 fusion protein
EP1987845B1 (fr) 1997-11-20 2012-03-21 Vical Incorporated Traitement contre le cancer utilisant des polynucléotides à expression cytokine et compositions correspondantes
WO2000015249A1 (fr) 1998-09-15 2000-03-23 Genetics Institute, Inc. Traitement de la maladie de kaposi avec il-12
ATE305801T1 (de) 2000-01-20 2005-10-15 Univ Zuerich Inst Fuer Medizin Intratumorale verabreichung nackter il-12 codierender nukleinsäuremoleküle
ES2164011B1 (es) 2000-02-25 2003-05-16 Inst Cientifico Tecnol Navarra Uso combinado de la quimiocina ip-10 y la interleucina-12 en la preparacion de composiciones para el tratamiento de tumores malignos.
AU3958101A (en) 2000-03-15 2001-09-24 Univ Pohang Genes of il-12p40 subunit mutated for improving the activity of il-12 and use thereof for dna vaccine adjuvant
KR20020010206A (ko) 2000-07-27 2002-02-04 이시우 인터루킨 12와 보조활성인자 ｂ7.1 유전자를 함유하는ｄｎａ 벡터 및 이벡터가 도입된 항암 세포백신
AU2002354799A1 (en) 2001-07-03 2003-01-21 University Of Utah Research Foundation Soluble steroidal peptides for nucleic acid delivery
US7595303B1 (en) 2002-09-05 2009-09-29 University Of South Florida Genetic adjuvants for immunotherapy
EA200501937A1 (ru) 2003-05-30 2006-08-25 Университет Южной Флориды Способ лечения злокачественных опухолей
GB0329146D0 (en) 2003-12-16 2004-01-21 Glaxosmithkline Biolog Sa Vaccine
DE102004042546A1 (de) 2004-09-02 2006-03-09 Curevac Gmbh Kombinationstherapie zur Immunstimulation
EP1640018A1 (fr) 2004-09-24 2006-03-29 Universität Zürich Thérapie combinatoire pour le traitement du cancer
ES2384055T3 (es) 2005-12-30 2012-06-28 Merck Patent Gmbh Variantes de la interleucina-12p40 con estabilidad mejorada
WO2007084364A2 (fr) 2006-01-13 2007-07-26 The Government Of The United States, As Represented By The Secretary Of Health And Human Services, National Institutes Of Health Il-12 améliorée aux fins d'expression dans des cellules mammaliennes
KR101241477B1 (ko) 2006-01-27 2013-03-08 엘지이노텍 주식회사 질화물 반도체 발광소자 및 그 제조 방법
DE102006051516A1 (de) 2006-10-31 2008-05-08 Curevac Gmbh (Basen-)modifizierte RNA zur Expressionssteigerung eines Proteins
EP3293266A1 (fr) 2007-05-04 2018-03-14 University Health Network Immunothérapie par il-12 pour le cancer
TWI359030B (en) 2007-08-24 2012-03-01 Univ Nat Taiwan Treatment of cellular proliferative disorders
PT2201100E (pt) 2007-09-14 2016-06-03 Univ Brussel Vrije Aperfeiçoamento da capacidade estimulatória de células t das células que apresentam antígeno humano e o seu uso na vacinação
WO2009140206A1 (fr) 2008-05-11 2009-11-19 The Gov. Of The U. S. A., As Represented By The Secretary Of The Dept. Of Health And Human Services Expression améliorée d'hétérodimères de la famille il-12
WO2009149539A1 (fr) 2008-06-10 2009-12-17 Université de Montréal Amplification de la réponse de lymphocytes t cd8+ spécifiques d’un antigène à l’aide d’arnm d’irf‑7
WO2010126766A1 (fr) 2009-04-30 2010-11-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Interleukine-12 inductible
AU2011358150B2 (en) 2010-12-16 2016-11-03 Sprna Gmbh Pharmaceutical composition consisting of RNA having alkali metal as counter ion and formulated with dications
WO2012116714A1 (fr) 2011-03-02 2012-09-07 Curevac Gmbh Vaccination chez des patients âgés
WO2013051672A1 (fr) 2011-10-04 2013-04-11 株式会社ヤクルト本社 Agent médicinal comprenant un dérivé de thiazolidine ou sel de celui-ci comme ingrédient actif
CA2850575C (fr) 2011-10-11 2020-12-15 Universitat Zurich Prorektorat Mnw Combinaison medicamenteuse comprenant de l'il-12 et un agent de blocage de molecules inhibitrices des lymphocytes t pour une therapie anti-tumorale
EP3336096A1 (fr) 2011-12-12 2018-06-20 The Trustees Of The University Of Pennsylvania Compositions comprenant des constructions génétiques d'il-12 améliorés et des vaccins améliorés, produits immunothérapeutiques et procédés d'utilisation associés
EP2623121A1 (fr) 2012-01-31 2013-08-07 Bayer Innovation GmbH Composition pharmaceutique comportant un complexe de chargement de porteur polymérique et un antigène
WO2014089486A1 (fr) 2012-12-07 2014-06-12 Shire Human Genetic Therapies, Inc. Nanoparticules lipidiques pour administration de marn
WO2014127917A1 (fr) 2013-02-22 2014-08-28 Curevac Gmbh Combinaison d'une vaccination et de l'inhibition de la voie de pd-1
JP6558699B2 (ja) 2013-10-01 2019-08-14 国立大学法人三重大学 抗原提示を促進するエピトープ間配列を含むｔ細胞誘導ワクチン
EP3058082A4 (fr) 2013-10-18 2017-04-26 ModernaTX, Inc. Compositions et procédés pour tolériser des systèmes cellulaires
EA201992208A1 (ru) 2013-10-22 2020-07-31 Транслейт Био, Инк. ЛЕЧЕНИЕ ФЕНИЛКЕТОНУРИИ С ПРИМЕНЕНИЕМ мРНК
WO2015095249A1 (fr) 2013-12-18 2015-06-25 Intrexon Corporation Acides nucléiques d'il-12 monocaténaires, polypeptides d'il-12 monocaténaires, et leurs utilisations
CA3012602A1 (fr) 2015-01-30 2016-08-04 President And Fellows Of Harvard College Materiaux peritumoraux et intratumoraux pour traitement anticancereux
SG11201707832RA (en) 2015-04-22 2017-11-29 Curevac Ag Rna containing composition for treatment of tumor diseases
US10682401B2 (en) 2015-05-19 2020-06-16 Morphogenesis, Inc. Multi-indication mRNA cancer immunotherapy
AU2016264363B2 (en) 2015-05-19 2019-07-25 Morphogenesis, Inc. Cancer vaccine comprising mRNA encoding a M-like-protein
HUE057613T2 (hu) 2015-09-17 2022-05-28 Modernatx Inc Vegyületek és készítmények terápiás szerek intracelluláris bejuttatására
EP3359670B2 (fr) 2015-10-05 2024-02-14 ModernaTX, Inc. Procédés d'administration thérapeutique de médicaments à base d'acide ribonucléique messager
EP3205356A1 (fr) 2016-02-10 2017-08-16 Onkoloski Institut Ljubljana Vecteur d'expression plasmidique codant l'interleukine humain 12 sous le contrôle transcriptionnel d'un promoteur p21 et sans la résistance aux antibiotiques pour la thérapie génique du cancer et d'autres utilisations
EP3211000B1 (fr) 2016-02-25 2019-01-30 Provecs Medical GmbH Nouveau système de vecteur immunostimulant
ES2941411T3 (es) 2016-05-18 2023-05-22 Modernatx Inc Polinucleótidos que codifican interleucina-12 (IL12) y usos de los mismos
SG10201913631TA (en) 2016-08-19 2020-03-30 Curevac Ag Rna for cancer therapy
CN110267978A (zh) 2016-10-07 2019-09-20 得克萨斯大学体系董事会 用于治疗癌症的表达膜锚定的il-12的t细胞
CA3063723A1 (fr) 2017-05-18 2018-11-22 Modernatx, Inc. Polynucleotides codant pour des polypeptides d'interleukine-12 (il12) ancres et leurs utilisations

Patent Citations (270)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5563250A (en) 1987-12-02 1996-10-08 Neorx Corporation Cleavable conjugates for the delivery and release of agents in native form
US5082767A (en) 1989-02-27 1992-01-21 Hatfield G Wesley Codon pair utilization
US5505931A (en) 1993-03-04 1996-04-09 The Dow Chemical Company Acid cleavable compounds, their preparation and use as bifunctional acid-labile crosslinking agents
US5681702A (en) 1994-08-30 1997-10-28 Chiron Corporation Reduction of nonspecific hybridization by using novel base-pairing schemes
US5780610A (en) 1994-08-30 1998-07-14 Collins; Mark L. Reduction of nonspecific hybridization by using novel base-pairing schemes
US5585108A (en) 1994-12-30 1996-12-17 Nanosystems L.L.C. Formulations of oral gastrointestinal therapeutic agents in combination with pharmaceutically acceptable clays
US20120178702A1 (en) 1995-01-23 2012-07-12 University Of Pittsburgh Stable lipid-comprising drug delivery complexes and methods for their production
US6265389B1 (en) 1995-08-31 2001-07-24 Alkermes Controlled Therapeutics, Inc. Microencapsulation and sustained release of oligonucleotides
US6555525B2 (en) 1995-08-31 2003-04-29 Alkermes Controlled Therapeutics, Inc. Microencapsulation and sustained release of oligonucleotides
US8404222B2 (en) 1996-09-26 2013-03-26 Nektar Therapeutics Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution
US20100036115A1 (en) 1997-07-23 2010-02-11 Sirna Therapeutics, Inc. Novel Compositions for the Delivery of Negatively Charged Molecules
US6004573A (en) 1997-10-03 1999-12-21 Macromed, Inc. Biodegradable low molecular weight triblock poly(lactide-co-glycolide) polyethylene glycol copolymers having reverse thermal gelation properties
US6267987B1 (en) 1997-12-12 2001-07-31 Samyang Corporation Positively charged poly[alpha-(omega-aminoalkyl) glycolic acid] for the delivery of a bioactive agent via tissue and cellular uptake
US6517869B1 (en) 1997-12-12 2003-02-11 Expression Genetics, Inc. Positively charged poly(alpha-(omega-aminoalkyl)lycolic acid) for the delivery of a bioactive agent via tissue and cellular uptake
US8003129B2 (en) 1998-01-05 2011-08-23 University Of Washington Enhanced transport using membrane disruptive agents
US7374778B2 (en) 1998-01-05 2008-05-20 University Of Washington Enhanced transport using membrane disruptive agents
US6835393B2 (en) 1998-01-05 2004-12-28 University Of Washington Enhanced transport using membrane disruptive agents
US6426086B1 (en) 1998-02-03 2002-07-30 The Regents Of The University Of California pH-sensitive, serum-stable liposomes
US6177274B1 (en) 1998-05-20 2001-01-23 Expression Genetics, Inc. Hepatocyte targeting polyethylene glyco-grafted poly-L-lysine polymeric gene carrier
US20130184453A1 (en) 1998-07-01 2013-07-18 California Institute Of Technology Linear cyclodextrin copolymers
US6217912B1 (en) 1998-07-13 2001-04-17 Expression Genetics, Inc. Polyester analogue of poly-L-lysine as a soluble, biodegradable gene delivery carrier
WO2000018778A1 (fr) 1998-09-29 2000-04-06 Phylos, Inc. Synthese d'acides nucleiques fondee sur une generation aleatoire de codons
US8309139B2 (en) 1999-02-26 2012-11-13 Novartis Vaccines And Diagnostics, Inc. Microemulsions with adsorbed macromolecules and microparticles
US20130195898A1 (en) 1999-02-26 2013-08-01 Novartis Vaccines And Diagnostics, Inc. Microemulsions with adsorbed macromolecules and microparticles
US20130195923A1 (en) 1999-02-26 2013-08-01 Novartis Vaccines And Diagnostics, Inc. Microemulsions with adsorbed macromolecules and microparticles
US8206749B1 (en) 1999-02-26 2012-06-26 Novartis Vaccines And Diagnostics, Inc. Microemulsions with adsorbed macromolecules and microparticles
US7138382B2 (en) 1999-06-07 2006-11-21 Mirus Bio Corporation Compositions and methods for drug delivery using pH sensitive molecules
US6984720B1 (en) 1999-08-24 2006-01-10 Medarex, Inc. Human CTLA-4 antibodies
US7737108B1 (en) 2000-01-07 2010-06-15 University Of Washington Enhanced transport using membrane disruptive agents
US20040142474A1 (en) 2000-09-14 2004-07-22 Expression Genetics, Inc. Novel cationic lipopolymer as a biocompatible gene delivery agent
US6696038B1 (en) 2000-09-14 2004-02-24 Expression Genetics, Inc. Cationic lipopolymer as biocompatible gene delivery agent
US20030073619A1 (en) 2000-09-14 2003-04-17 Mahato Ram I. Novel cationic lipopolymer as biocompatible gene delivery agent
US20120228565A1 (en) 2000-10-13 2012-09-13 Life Technologies Corporation Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US7098032B2 (en) 2001-01-02 2006-08-29 Mirus Bio Corporation Compositions and methods for drug delivery using pH sensitive molecules
US6897196B1 (en) 2001-02-07 2005-05-24 The Regents Of The University Of California pH sensitive lipids based on ortho ester linkers, composition and method
US6652886B2 (en) 2001-02-16 2003-11-25 Expression Genetics Biodegradable cationic copolymers of poly (alkylenimine) and poly (ethylene glycol) for the delivery of bioactive agents
US20050032730A1 (en) 2001-06-05 2005-02-10 Florian Von Der Mulbe Pharmaceutical composition containing a stabilised mRNA optimised for translation in its coding regions
US6586524B2 (en) 2001-07-19 2003-07-01 Expression Genetics, Inc. Cellular targeting poly(ethylene glycol)-grafted polymeric gene carrier
US20040262223A1 (en) 2001-07-27 2004-12-30 President And Fellows Of Harvard College Laminar mixing apparatus and methods
US20050059005A1 (en) 2001-09-28 2005-03-17 Thomas Tuschl Microrna molecules
US8257745B2 (en) 2001-12-21 2012-09-04 Novartis Ag Use of synthetic inorganic nanoparticles as carriers for ophthalmic and otic drugs
US20050222064A1 (en) 2002-02-20 2005-10-06 Sirna Therapeutics, Inc. Polycationic compositions for cellular delivery of polynucleotides
US8460709B2 (en) 2002-03-13 2013-06-11 Novartis Ag Pharmaceutical microparticles
US20080076161A1 (en) 2002-04-01 2008-03-27 The Government Of The United States, As Represented By The Secretary Of The Army Method of designing synthetic nucleic acid sequences for optimal protein expression in a host cell
US20120201859A1 (en) 2002-05-02 2012-08-09 Carrasquillo Karen G Drug Delivery Systems and Use Thereof
US20140228558A1 (en) 2002-12-23 2014-08-14 Geneart Ag Method and device for optimizing a nucelotide sequence for the purpose of expression in a protein
US8524368B2 (en) 2003-07-09 2013-09-03 Wisconsin Alumni Research Foundation Charge-dynamic polymers and delivery of anionic compounds
US20130177587A1 (en) 2003-07-11 2013-07-11 Novavax, Inc. Functional influenza virus-like particles (vlps)
US8506967B2 (en) 2003-07-11 2013-08-13 Novavax, Inc. Functional influenza virus like particles (VLPs)
US20050261218A1 (en) 2003-07-31 2005-11-24 Christine Esau Oligomeric compounds and compositions for use in modulation small non-coding RNAs
US8205511B1 (en) 2003-10-17 2012-06-26 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Air-sampling device and method of use
US8236280B2 (en) 2003-12-19 2012-08-07 University Of Cincinnati Polyamides for nucleic acid delivery
US8241670B2 (en) 2004-04-15 2012-08-14 Chiasma Inc. Compositions capable of facilitating penetration across a biological barrier
US20130177611A1 (en) 2004-06-11 2013-07-11 Eidgenossisches Technische Hochschule (The Swiss Federal Institute of Technology) Silk-based drug delivery system
US8057821B2 (en) 2004-11-03 2011-11-15 Egen, Inc. Biodegradable cross-linked cationic multi-block copolymers for gene delivery and methods of making thereof
US20120009145A1 (en) 2004-11-03 2012-01-12 Gregory Slobodkin Biodegradable Cross-Linked Cationic Multi-block Copolymers for Gene Delivery and Methods of Making Thereof
WO2006063249A2 (fr) 2004-12-10 2006-06-15 Justin Hanes Copolymeres sequences de poly(ether-anhydride) fonctionnalises
US7404969B2 (en) 2005-02-14 2008-07-29 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
US7893302B2 (en) 2005-02-14 2011-02-22 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
US20130059360A1 (en) 2005-04-12 2013-03-07 Nektar Therapeutics Polymer-based compositions and conjugates of antimicrobial agents
US8008449B2 (en) 2005-05-09 2011-08-30 Medarex, Inc. Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics
WO2007024323A2 (fr) 2005-06-17 2007-03-01 The University Of North Carolina At Chapel Hill Procedes, systemes et materiaux de fabrication de nanoparticules
US8101385B2 (en) 2005-06-30 2012-01-24 Archemix Corp. Materials and methods for the generation of transcripts comprising modified nucleotides
US20070117112A1 (en) 2005-06-30 2007-05-24 Diener John L Materials and methods for the generation of fully 2'-modified nucleic acid transcripts
US20100120024A1 (en) 2005-06-30 2010-05-13 Sharon Cload Materials and methods for the generation of transcripts comprising modified nucleotides
US7943743B2 (en) 2005-07-01 2011-05-17 Medarex, Inc. Human monoclonal antibodies to programmed death ligand 1 (PD-L1)
US8444992B2 (en) 2005-09-01 2013-05-21 Novartis Vaccines And Diagnostics Gmbh Multiple vaccination including serogroup C meningococcus
US20100129877A1 (en) 2005-09-28 2010-05-27 Ugur Sahin Modification of RNA, Producing an Increased Transcript Stability and Translation Efficiency
US20130137644A1 (en) 2005-12-16 2013-05-30 Cellectis Cell penetrating peptide conjugates for delivering of nucleic acids into a cell
US20130129726A1 (en) 2006-02-20 2013-05-23 Kyunglim Lee Peptide having cell membrane penetrating activity
US20130072709A1 (en) 2006-02-21 2013-03-21 Nektar Therapeutics Segmented Degradable Polymers and Conjugates Made Therefrom
WO2007113648A2 (fr) 2006-04-05 2007-10-11 Pfizer Products Inc. Polythérapie à base d'un anticorps anti-ctla4
US20130130348A1 (en) 2006-05-15 2013-05-23 The Brigham And Women's Hospital, Inc. Polymers for Functional Particles
US20080046192A1 (en) 2006-08-16 2008-02-21 Richard Lathrop Polypepetide-encoding nucleotide sequences with refined translational kinetics and methods of making same
US8313777B2 (en) 2006-10-05 2012-11-20 The Johns Hopkins University Water-dispersible oral, parenteral, and topical formulations for poorly water soluble drugs using smart polymeric nanoparticles
US8524259B2 (en) 2006-12-05 2013-09-03 Landec Corporation Systems and methods for delivery of materials
US20130150295A1 (en) 2006-12-21 2013-06-13 Stryker Corporation Sustained-Release Formulations Comprising Crystals, Macromolecular Gels, and Particulate Suspensions of Biologic Agents
WO2008078180A2 (fr) 2006-12-22 2008-07-03 Archemix Corp. Matériaux et procédés pour la génération de transcrits comprenant des nucléotides modifiés
US8507653B2 (en) 2006-12-27 2013-08-13 Nektar Therapeutics Factor IX moiety-polymer conjugates having a releasable linkage
WO2008103276A2 (fr) 2007-02-16 2008-08-28 Merck & Co., Inc. Compositions et méthodes de potentialisation de l'activité de molécules biologiquement actives
US8496945B2 (en) 2007-03-05 2013-07-30 Washington University Nanoparticle delivery systems for membrane-integrating peptides
US8246968B2 (en) 2007-03-30 2012-08-21 Bind Biosciences, Inc. Cancer cell targeting using nanoparticles
WO2008121949A1 (fr) 2007-03-30 2008-10-09 Bind Biosciences, Inc. Ciblage de cellules cancéreuses utilisant des nanoparticules
US8501824B2 (en) 2007-05-04 2013-08-06 Marina Biotech, Inc. Amino acid lipids and uses thereof
US8900587B2 (en) 2007-06-18 2014-12-02 Merck Sharp & Dohme Corp. Antibodies to human programmed death receptor PD-1
US20100293625A1 (en) 2007-09-26 2010-11-18 Interexon Corporation Synthetic 5'UTRs, Expression Vectors, and Methods for Increasing Transgene Expression
US8236330B2 (en) 2007-09-28 2012-08-07 Bind Biosciences, Inc. Cancer cell targeting using nanoparticles
US20130172406A1 (en) 2007-09-28 2013-07-04 Bind Biosciences, Inc. Cancer Cell Targeting Using Nanoparticles
US20120004293A1 (en) 2007-09-28 2012-01-05 Zale Stephen E Cancer Cell Targeting Using Nanoparticles
US20090226470A1 (en) 2007-12-11 2009-09-10 Mauro Vincent P Compositions and methods related to mRNA translational enhancer elements
US20100004315A1 (en) 2008-03-14 2010-01-07 Gregory Slobodkin Biodegradable Cross-Linked Branched Poly(Alkylene Imines)
US8519110B2 (en) 2008-06-06 2013-08-27 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College mRNA cap analogs
US8401798B2 (en) 2008-06-06 2013-03-19 Dna Twopointo, Inc. Systems and methods for constructing frequency lookup tables for expression systems
US8318211B2 (en) 2008-06-16 2012-11-27 Bind Biosciences, Inc. Therapeutic polymeric nanoparticles comprising vinca alkaloids and methods of making and using same
US8318208B1 (en) 2008-06-16 2012-11-27 Bind Biosciences, Inc. Drug loaded polymeric nanoparticles and methods of making and using same
US8293276B2 (en) 2008-06-16 2012-10-23 Bind Biosciences, Inc. Drug loaded polymeric nanoparticles and methods of making and using same
WO2010005723A2 (fr) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Nanoparticules polymères pharmacologiquement chargées et leurs méthodes de fabrication et d’utilisation
US20100069426A1 (en) 2008-06-16 2010-03-18 Zale Stephen E Therapeutic polymeric nanoparticles with mTor inhibitors and methods of making and using same
US20130230567A1 (en) 2008-06-16 2013-09-05 Bind Therapeutics, Inc. Drug Loaded Polymeric Nanoparticles and Methods of Making and Using Same
WO2010005725A2 (fr) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Nanoparticules polymères thérapeutiques comprenant des alcaloïdes vinca et procédés de fabrication et d’utilisation associés
US20120288541A1 (en) 2008-06-16 2012-11-15 Zale Stephen E Drug Loaded Polymeric Nanoparticles and Methods of Making and Using Same
WO2010005721A2 (fr) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Nanoparticules polymères pharmacologiquement chargées et leurs méthodes de fabrication et d’utilisation
US20100068285A1 (en) 2008-06-16 2010-03-18 Zale Stephen E Drug Loaded Polymeric Nanoparticles and Methods of Making and Using Same
WO2010005740A2 (fr) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Procédés pour la préparation de copolymères diblocs fonctionnalisés avec un agent de ciblage destinés à être utilisés dans la fabrication de nanoparticules ciblées thérapeutiques
US20100104655A1 (en) 2008-06-16 2010-04-29 Zale Stephen E Therapeutic Polymeric Nanoparticles Comprising Vinca Alkaloids and Methods of Making and Using Same
WO2010005726A2 (fr) 2008-06-16 2010-01-14 Bind Biosciences Inc. Nanoparticules polymères thérapeutiques avec inhibiteurs de mtor et procédés de fabrication et d’utilisation associés
US8206747B2 (en) 2008-06-16 2012-06-26 Bind Biosciences, Inc. Drug loaded polymeric nanoparticles and methods of making and using same
US20100104645A1 (en) 2008-06-16 2010-04-29 Bind Biosciences, Inc. Methods for the preparation of targeting agent functionalized diblock copolymers for use in fabrication of therapeutic targeted nanoparticles
US20100068286A1 (en) 2008-06-16 2010-03-18 Greg Troiano Drug Loaded Polymeric Nanoparticles and Methods of Making and Using Same
US20110274759A1 (en) 2008-06-16 2011-11-10 Greg Troiano Drug Loaded Polymeric Nanoparticles and Methods of Making and Using Same
US20100009424A1 (en) 2008-07-14 2010-01-14 Natasha Forde Sonoporation systems and methods
US8126653B2 (en) 2008-07-31 2012-02-28 Dna Twopointo, Inc. Synthetic nucleic acids for expression of encoded proteins
US7561973B1 (en) 2008-07-31 2009-07-14 Dna Twopointo, Inc. Methods for determining properties that affect an expression property value of polynucleotides in an expression system
US20110224447A1 (en) 2008-08-18 2011-09-15 Bowman Keith A Novel Lipid Nanoparticles and Novel Components for Delivery of Nucleic Acids
WO2010021865A1 (fr) 2008-08-18 2010-02-25 Merck Sharp & Dohme Corp. Nouvelles nanoparticules lipidiques et nouveaux composants pour l'administration d'acides nucléiques
US8609089B2 (en) 2008-08-25 2013-12-17 Amplimmune, Inc. Compositions of PD-1 antagonists and methods of use
WO2010030763A2 (fr) 2008-09-10 2010-03-18 Bind Biosciences, Inc. Fabrication de nanoparticles à rendement élevé
US20130123351A1 (en) 2008-09-10 2013-05-16 Bind Biosciences, Inc. High throughput fabrication of nanoparticles
US20100087337A1 (en) 2008-09-10 2010-04-08 Bind Biosciences, Inc. High Throughput Fabrication of Nanoparticles
CN101684456A (zh) * 2008-09-28 2010-03-31 江门罗森生物制药有限公司 一种体外培养条件下扩增人nk细胞的方法
US20120015899A1 (en) 2008-10-25 2012-01-19 Plant Bioscience, Limited Modified plant virus particles and uses therefor
WO2010047839A1 (fr) 2008-10-25 2010-04-29 Aura Biosciences Particules de virus de plante modifiées et utilisations de celles-ci
US8450298B2 (en) 2008-11-07 2013-05-28 Massachusetts Institute Of Technology Aminoalcohol lipidoids and uses thereof
US8217149B2 (en) 2008-12-09 2012-07-10 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
US20110217377A1 (en) 2008-12-15 2011-09-08 Zale Stephen E Long Circulating Nanoparticles for Sustained Release of Therapeutic Agents
US20100216804A1 (en) 2008-12-15 2010-08-26 Zale Stephen E Long Circulating Nanoparticles for Sustained Release of Therapeutic Agents
WO2010075072A2 (fr) 2008-12-15 2010-07-01 Bind Biosciences Longues nanoparticules circulantes pour la libération prolongée d'agents thérapeutiques
WO2010080724A1 (fr) 2009-01-12 2010-07-15 Merck Sharp & Dohme Corp. Nanoparticules lipidiques inédites et composants inédits utilisables pour l'administration d'acides nucléiques
WO2010087791A1 (fr) 2009-01-27 2010-08-05 Utc Power Corporation Réacteur de conversion à la vapeur d'eau intégré, refroidi de manière distributive et atomiseur
US20100196983A1 (en) 2009-02-05 2010-08-05 Ut-Battelle, Llc Transformation of gram positive bacteria by sonoporation
US8460696B2 (en) 2009-03-20 2013-06-11 Egen, Inc. Polyamine derivatives
WO2010120266A1 (fr) 2009-04-13 2010-10-21 Inserm, Institut National De La Sante Et De La Recherche Medicale Particules hpv et utilisations associées
US20120171290A1 (en) 2009-04-13 2012-07-05 Coursaget Pierre L Hpv particles and uses thereof
US20100324120A1 (en) 2009-06-10 2010-12-23 Jianxin Chen Lipid formulation
US8158601B2 (en) 2009-06-10 2012-04-17 Alnylam Pharmaceuticals, Inc. Lipid formulation
US20140121263A1 (en) 2009-06-15 2014-05-01 Alnylam Pharmaceuticals, Inc. Lipid formulated dsrna targeting the pcsk9 gene
US8283333B2 (en) 2009-07-01 2012-10-09 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
US20120202871A1 (en) 2009-07-01 2012-08-09 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
WO2011022460A1 (fr) 2009-08-20 2011-02-24 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques avec différents groupes de tête pour délivrance d’oligonucléotide
WO2011043913A2 (fr) 2009-10-08 2011-04-14 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques à chaînes lipidiques courtes pour une administration d'oligonucléotides
US20120276209A1 (en) 2009-11-04 2012-11-01 The University Of British Columbia Nucleic acid-containing lipid particles and related methods
WO2011062965A2 (fr) 2009-11-18 2011-05-26 University Of Washington Through Its Center For Commercialization Monomères de ciblage et polymère ayant des blocs de ciblage
US20130017199A1 (en) 2009-11-24 2013-01-17 AMPLIMMUNE ,Inc. a corporation Simultaneous inhibition of pd-l1/pd-l2
US20120258046A1 (en) 2009-12-09 2012-10-11 Thorsten Mutzke Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids
WO2011084521A2 (fr) 2009-12-15 2011-07-14 Bind Biosciences, Inc. Nanoparticules polymères thérapeutiques comportant de l'épothilone et leurs procédés de fabrication et d'utilisation
WO2011084518A2 (fr) 2009-12-15 2011-07-14 Bind Biosciences, Inc. Nanoparticules polymères thérapeutiques comprenant de corticostéroïdes, et procédés pour les fabriquer et les utiliser
US20120140790A1 (en) 2009-12-15 2012-06-07 Ali Mir M Therapeutic Polymeric Nanoparticle Compositions with High Glass Transition Termperature or High Molecular Weight Copolymers
WO2011085231A2 (fr) 2010-01-08 2011-07-14 Selecta Biosciences, Inc. Pseudo-particules virales synthétiques conjuguées à des peptides de capside de papillomavirus humain pour une utilisation en tant que vaccins
US20110171248A1 (en) 2010-01-08 2011-07-14 Selecta Biosciences, Inc. Synthetic virus-like particles conjugated to human papillomavirus capsid peptides for use as vaccines
WO2011090965A1 (fr) 2010-01-22 2011-07-28 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques pour transfert d'oligonucléotide
US20130231287A1 (en) 2010-02-25 2013-09-05 Parimala Nacharaju Pegylated albumin polymers and uses thereof
WO2011127255A1 (fr) 2010-04-08 2011-10-13 Merck Sharp & Dohme Corp. Préparation de nanoparticules de lipide
US20130177636A1 (en) 2010-04-09 2013-07-11 Pacira Pharmaceuticals, Inc. Method for formulating large diameter synthetic membrane vesicles
US20130177633A1 (en) 2010-04-09 2013-07-11 Pacira Pharmaceuticals, Inc. Method for formulating large diameter synthetic membrane vesicles
US20130183375A1 (en) 2010-04-09 2013-07-18 Pacira Pharmaceuticals, Inc. Method for formulating large diameter synthetic membrane vesicles
US20130177638A1 (en) 2010-04-09 2013-07-11 Pacira Pharmaceuticals, Inc. Method for formulating large diameter synthetic membrane vesicles
US20130177637A1 (en) 2010-04-09 2013-07-11 Pacira Pharmaceuticals, Inc. Method for formulating large diameter synthetic membrane vesicles
US20130183373A1 (en) 2010-04-09 2013-07-18 Pacira Pharmaceuticals, Inc. Method for formulating large diameter synthetic membrane vesicles
US20130177634A1 (en) 2010-04-09 2013-07-11 Pacira Pharmaceuticals, Inc. Method for formulating large diameter synthetic membrane vesicles
US20130183372A1 (en) 2010-04-09 2013-07-18 Pacira Pharmaceuticals, Inc. Method for formulating large diameter synthetic membrane vesicles
US20130177635A1 (en) 2010-04-09 2013-07-11 Pacira Pharmaceuticals, Inc. Method for formulating large diameter synthetic membrane vesicles
US20110262491A1 (en) 2010-04-12 2011-10-27 Selecta Biosciences, Inc. Emulsions and methods of making nanocarriers
US20130129785A1 (en) 2010-05-10 2013-05-23 Alnylam Pharmaceuticals, Inc Methods and compositions for delivery of active agents
US20130123338A1 (en) 2010-05-12 2013-05-16 Protiva Biotherapeutics, Inc. Novel cationic lipids and methods of use thereof
WO2011149733A2 (fr) 2010-05-24 2011-12-01 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques alcools aminés pour l'administration d'oligonucléotides
US20130150625A1 (en) 2010-05-24 2013-06-13 Brian W. Budzik Novel Amino Alcohol Cationic Lipids for Oligonucleotide Delivery
US20130209544A1 (en) 2010-05-26 2013-08-15 Micromedmark Biotech Co., Ltd. Microvesicles carrying small interfering rnas, preparation methods and uses thereof
US20150315112A1 (en) 2010-06-04 2015-11-05 Sirna Therapeutics, Inc. Novel low molecular weight cationic lipids for oligonucleotide delivery
WO2011153120A1 (fr) 2010-06-04 2011-12-08 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques de faible poids moléculaire pour l'administration d'oligonucléotides
US20130164219A1 (en) 2010-06-14 2013-06-27 Hoffmann-La Roche Inc. Cell-penetrating peptides and uses thereof
US20130196948A1 (en) 2010-06-25 2013-08-01 Massachusetts Insitute Of Technology Polymers for biomaterials and therapeutics
US20130171241A1 (en) 2010-07-06 2013-07-04 Novartis Ag Liposomes with lipids having an advantageous pka-value for rna delivery
WO2012006376A2 (fr) 2010-07-06 2012-01-12 Novartis Ag Particules d'administration de type virion pour des molécules d'arn auto-répliquant
WO2012006378A1 (fr) 2010-07-06 2012-01-12 Novartis Ag Liposomes à lipides ayant une valeur de pka avantageuse pour la délivrance d'arn
US20130195968A1 (en) 2010-07-06 2013-08-01 Novartis Ag Virion-like delivery particles for self-replicating rna molecules
WO2012006380A2 (fr) 2010-07-06 2012-01-12 Novartis Ag Émulsions cationiques huile-dans-eau
US20130211249A1 (en) 2010-07-22 2013-08-15 The Johns Hopkins University Drug eluting hydrogels for catheter delivery
US20130142818A1 (en) 2010-07-30 2013-06-06 Curevac Gmbh Complexation of nucleic acids with disulfide-crosslinked cationic components for transfection and immunostimulation
WO2012013326A1 (fr) 2010-07-30 2012-02-02 Curevac Gmbh Complexation d'acides nucléiques avec des composants cationiques réticulés par un pont disulfure pour une transfection et une immunostimulation
US20130171646A1 (en) 2010-08-09 2013-07-04 So Jung PARK Nanop article-oligonucleotide hybrid structures and methods of use thereof
US20130195799A1 (en) 2010-08-19 2013-08-01 Peg Biosciences, Inc. Synergistic biomolecule-polymer conjugates
WO2012031046A2 (fr) 2010-08-31 2012-03-08 Novartis Ag Lipides adaptés pour une administration liposomale d'arn codant pour une protéine
US20130195969A1 (en) 2010-08-31 2013-08-01 Novartis Ag Small liposomes for delivery of immunogen encoding rna
WO2012030901A1 (fr) 2010-08-31 2012-03-08 Novartis Ag Petits liposomes destinés à l'administration d'un arn codant pour un immunogène
US20130189351A1 (en) 2010-08-31 2013-07-25 Novartis Ag Lipids suitable for liposomal delivery of protein coding rna
WO2012031043A1 (fr) 2010-08-31 2012-03-08 Novartis Ag Liposomes pégylés pour l'apport d'arn codant pour un immunogène
US20130202684A1 (en) 2010-08-31 2013-08-08 Lichtstrasse Pegylated liposomes for delivery of immunogen encoding rna
US20130183244A1 (en) 2010-09-10 2013-07-18 The Johns Hopkins University Rapid Diffusion of Large Polymeric Nanoparticles in the Mammalian Brain
US8466122B2 (en) 2010-09-17 2013-06-18 Protiva Biotherapeutics, Inc. Trialkyl cationic lipids and methods of use thereof
WO2012040184A2 (fr) 2010-09-20 2012-03-29 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques de faible poids moléculaire pour l'administration d'oligonucléotides
US20130178541A1 (en) 2010-09-20 2013-07-11 Matthew G. Stanton Novel low molecular weight cationic lipids for oligonucleotide delivery
WO2012040524A1 (fr) 2010-09-24 2012-03-29 Mallinckrodt Llc Conjugués d'aptamères pour ciblage de nanovecteurs thérapeutiques et/ou diagnostiques
WO2012044638A1 (fr) 2010-09-30 2012-04-05 Merck Sharp & Dohme Corp. Lipides cationiques de faible masse moléculaire utilisables en vue de l'administration d'oligonucléotides
US20120265001A1 (en) 2010-10-11 2012-10-18 Wichita State University Composite magnetic nanoparticle drug delivery system
WO2012049366A1 (fr) 2010-10-14 2012-04-19 Timo Vesikari Protéine de capside de norovirus et de rotavirus vp6 a usage comme vaccin combine
WO2012054365A2 (fr) 2010-10-21 2012-04-26 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques à faible poids moléculaire destinés à une administration d'oligonucléotides
WO2012054923A2 (fr) 2010-10-22 2012-04-26 Bind Biosciences, Inc. Nanoparticules thérapeutiques contenant des copolymères de masse moléculaire élevée
US20130225836A1 (en) 2010-11-05 2013-08-29 Merck Sharp & Dohme Corp. Novel low molecular weight cyclic amine containing cationic lipids for oligonucleotide delivery
WO2012061259A2 (fr) 2010-11-05 2012-05-10 Merck Sharp & Dohme Corp. Amine cyclique inédite de faible masse moléculaire contenant des lipides cationiques en vue de l'administration d'oligonucléotides
WO2012109121A1 (fr) 2011-02-07 2012-08-16 Purdue Research Foundation Nanoparticules glucidiques pour une efficacité prolongée d'un peptide antimicrobien
US20120207840A1 (en) 2011-02-10 2012-08-16 Aura Biosciences, Inc. Virion Derived Protein Nanoparticles For Delivering Diagnostic Or Therapeutic Agents For The Treatment Of Non-Melanoma Skin Cancer
WO2012110636A2 (fr) 2011-02-18 2012-08-23 Instituto Nacional De Investigación Y Tecnología Agraria Y Alimentaria (Inia) Peptides supports pour distribution dans cellules
US20130217753A1 (en) 2011-02-22 2013-08-22 Rutgers, The State University Of New Jersey Amphiphilic macromolecules for nucleic acid delivery
US20120263677A1 (en) 2011-03-10 2012-10-18 Provectus Pharmaceuticals, Inc. Combination of Local and Systemic Immunomodulative Therapies for Enhanced Treatment of Cancer
WO2012122444A1 (fr) 2011-03-10 2012-09-13 Provectus Pharmaceuticals, Inc. Combinaison de thérapies immunomodulatrices locales et systémiques pour l'amélioration du traitement du cancer
WO2012135805A2 (fr) 2011-03-31 2012-10-04 modeRNA Therapeutics Administration et formulation d'acides nucléiques génétiquement modifiés
US8710200B2 (en) 2011-03-31 2014-04-29 Moderna Therapeutics, Inc. Engineered nucleic acids encoding a modified erythropoietin and their expression
US20120283503A1 (en) 2011-04-29 2012-11-08 The Johns Hopkins University Nanoparticle loaded stem cells and their use in mri guided hyperthermia
US20120295832A1 (en) 2011-05-17 2012-11-22 Arrowhead Research Corporation Novel Lipids and Compositions for Intracellular Delivery of Biologically Active Compounds
WO2012170930A1 (fr) 2011-06-08 2012-12-13 Shire Human Genetic Therapies, Inc Compositions de nanoparticules lipides et procédés pour le transfert d'arnm
WO2013007604A1 (fr) 2011-07-08 2013-01-17 Bayer Intellectual Property Gmbh Procédé de production de dérivés de diamide d'acide anthranilique substitués par tétrazole par mise en réaction d'acides pyrazoliques avec des esters d'acide anthranilique
US20130012450A1 (en) 2011-07-10 2013-01-10 Aura Biosciences, Inc. Virion Derived Protein Nanoparticles For Delivering Diagnostic Or Therapeutic Agents For The Treatment Of Dermatology Related Genetic Diseases
US20130012426A1 (en) 2011-07-10 2013-01-10 Aura Biosciences, Inc. Virion Derived Protein Nanoparticles For Delivering Diagnostic Or Therapeutic Agents For the Treatment of Psoriasis
US20130012566A1 (en) 2011-07-10 2013-01-10 Aura Biosciences, Inc. Virion Derived Protein Nanoparticles For Delivering Diagnostic Or Therapeutic Agents For The Treatment of Alopecia
WO2013009717A1 (fr) 2011-07-10 2013-01-17 Elisabet De Los Pinos Nanoparticules de protéine virionique pour l'administration d'agents diagnostiques ou thérapeutiques pour le traitement de maladies cutanées
US20130064894A1 (en) 2011-08-31 2013-03-14 Protiva Biotherapeutics, Inc. Novel cationic lipids and methods of use thereof
WO2013033438A2 (fr) 2011-08-31 2013-03-07 Mallinckrodt Llc Modification de nanoparticules de peg avec des h-phosphonates
WO2013039857A1 (fr) 2011-09-12 2013-03-21 modeRNA Therapeutics Acides nucléiques modifiés et leurs procédés d'utilisation
WO2013052523A1 (fr) 2011-10-03 2013-04-11 modeRNA Therapeutics Nucléosides, nucléotides et acides nucléiques modifiés, et leurs utilisations
US20130115272A1 (en) 2011-10-03 2013-05-09 modeRNA Therapeutics Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US20130115247A1 (en) 2011-11-05 2013-05-09 Aura Biosciences, Inc. Virion Derived Protein Nanoparticles For Delivering Radioisotopes For The Diagnosis And Treatment Of Malignant And Systemic Disease And The Monitoring Of Therapy
US20130116408A1 (en) 2011-11-05 2013-05-09 Aura Biosciences, Inc. Virion Derived Protein Nanoparticles For Delivering Radioisotopes For The Diagnosis And Treatment Of Malignant And Systemic Disease And The Monitoring Of Therapy
US20130129830A1 (en) 2011-11-18 2013-05-23 Regeneron Pharmaceuticals, Inc. Polymer Protein Microparticles
WO2013075068A1 (fr) 2011-11-18 2013-05-23 Regeneron Pharmaceuticals, Inc. Microparticules de protéine et de polymère
US20140341917A1 (en) 2011-11-28 2014-11-20 Merck Patent Gmbh Anti-pd-l1 antibodies and uses thereof
WO2013082111A2 (fr) 2011-11-29 2013-06-06 The University Of North Carolina At Chapel Hill Particules manipulées de façon géométrique et procédés de modulation de réponses des macrophages ou immunitaires
WO2013084000A2 (fr) 2011-12-07 2013-06-13 Isis Innovation Limited Exosomes pour l'administration de biothérapiques
US20130195920A1 (en) 2011-12-07 2013-08-01 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
WO2013086322A1 (fr) 2011-12-07 2013-06-13 Alnylam Pharmaceuticals, Inc. Lipides biodégradables ramifiés à terminaisons alkyle et cycloalkyle destinés à l'administration d'agents actifs
WO2013086373A1 (fr) 2011-12-07 2013-06-13 Alnylam Pharmaceuticals, Inc. Lipides pour l'administration d'agents actifs
WO2013086526A1 (fr) 2011-12-09 2013-06-13 The Regents Of The University Of California Encapsulation liposomale de médicaments
WO2013088250A1 (fr) 2011-12-13 2013-06-20 Engeneic Molecular Delivery Pty Ltd Minicellules intactes, d'origine bactérienne, pour l'administration d'agents thérapeutiques à des tumeurs cérébrales
US20130177499A1 (en) 2011-12-13 2013-07-11 Engenelc Molecular Delivery Pty Ltd Bacterially derived, intact minicells for delivery of therapeutic agents to brain tumors
WO2013090648A1 (fr) 2011-12-16 2013-06-20 modeRNA Therapeutics Nucléoside, nucléotide, et compositions d'acide nucléique modifiés
US20130245107A1 (en) 2011-12-16 2013-09-19 modeRNA Therapeutics Dlin-mc3-dma lipid nanoparticle delivery of modified polynucleotides
WO2013106086A1 (fr) 2012-01-10 2013-07-18 Sorbent Therapeutics, Inc. Compositions comprenant des polymères réticulés de liaison à des cations et utilisations associées
WO2013106073A1 (fr) 2012-01-10 2013-07-18 Sorbent Therapeutics, Inc. Compositions comprenant des polymères réticulés de liaison à des cations et utilisations associées
WO2013106072A1 (fr) 2012-01-10 2013-07-18 Sorbent Therapeutics, Inc. Compositions comprenant des polymères réticulés de liaison à des cations et utilisations associées
WO2013106525A1 (fr) 2012-01-12 2013-07-18 Stc.Unm Vlp immunogènes contenant le hpv l2 et compositions et procédés associés
WO2013105101A1 (fr) 2012-01-13 2013-07-18 Department Of Biotechnology Nanoparticules lipidiques solides encapsulant un médicament hydrophile/amphiphile et leur procédé de préparation
WO2013110028A1 (fr) 2012-01-19 2013-07-25 The Johns Hopkins University Formulations de nanoparticules présentant une pénétration améliorée dans les muqueuses
WO2013116656A1 (fr) 2012-02-03 2013-08-08 Emory University Compositions immunostimulatrices, particules et applications associées
WO2013119602A1 (fr) 2012-02-06 2013-08-15 President And Fellows Of Harvard College Microvésicules médiées par arrdc1 (armm) et leurs utilisations
WO2013119877A1 (fr) 2012-02-07 2013-08-15 Aura Biosciences, Inc. Nanosphères dérivées de virions pour l'administration sélective d'agents thérapeutiques et diagnostiques dans des cellules cancéreuses
WO2013122262A1 (fr) 2012-02-16 2013-08-22 Vlp Therapeutics, Llc Composition à base d'une particule de type viral
WO2013123298A1 (fr) 2012-02-17 2013-08-22 University Of Georgia Research Foundation, Inc. Nanoparticules de trafic mitochondrial d'agents
WO2013123523A1 (fr) 2012-02-19 2013-08-22 Nvigen, Inc. Utilisations d'une nanostructure poreuse lors de son administration
WO2013124867A1 (fr) 2012-02-21 2013-08-29 Amrita Vishwa Vidyapeetham University Polymer - polymer or polymer - protein core - shell nano medicine loaded with multiple drug molecules
WO2013126803A1 (fr) 2012-02-24 2013-08-29 Protiva Biotherapeutics Inc. Lipides cationiques trialkylés et leurs procédés d'utilisation
WO2013151671A1 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés pour la production de protéines et de peptides cosmétiques
WO2013151666A2 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés destinés à la production de produits biologiques et de protéines associées à une maladie humaine
US20130259923A1 (en) 2012-04-02 2013-10-03 modeRNA Therapeutics Modified polynucleotides for the production of secreted proteins
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
WO2013173223A1 (fr) 2012-05-15 2013-11-21 Bristol-Myers Squibb Company Immunothérapie anticancéreuse par rupture de la signalisation pd-1/pd-l1
WO2014028429A2 (fr) 2012-08-14 2014-02-20 Moderna Therapeutics, Inc. Enzymes et polymérases destinées à la synthèse d'arn
US20150307542A1 (en) 2012-10-03 2015-10-29 Moderna Therapeutics, Inc. Modified nucleic acid molecules and uses thereof
WO2014093924A1 (fr) 2012-12-13 2014-06-19 Moderna Therapeutics, Inc. Molécules d'acide nucléique modifiées et leurs utilisations
US20140200261A1 (en) 2013-01-17 2014-07-17 Moderna Therapeutics, Inc. Signal-sensor polynucleotides for the alteration of cellular phenotypes
WO2014164253A1 (fr) 2013-03-09 2014-10-09 Moderna Therapeutics, Inc. Régions non traduites hétérologues pour arnm
WO2014152211A1 (fr) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation et administration de compositions de nucléosides, de nucléotides, et d'acides nucléiques modifiés
US20140363493A1 (en) 2013-06-10 2014-12-11 Albert Einstein College Of Medicine Of Yeshiva University LIPID NANOPARTICLES FOR TARGETED siRNA DELIVERY
WO2015011633A1 (fr) 2013-07-23 2015-01-29 Protiva Biotherapeutics, Inc. Compositions et procédés pour l'administration d'arn messager
US20150079109A1 (en) 2013-09-13 2015-03-19 Beigene, Ltd. Anti-PD1 Antibodies and their Use as Therapeutics and Diagnostics
WO2015051214A1 (fr) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucléotides codant pour un récepteur de lipoprotéines de faible densité
WO2015123264A1 (fr) 2014-02-11 2015-08-20 Alnylam Pharmaceuticals, Inc. Compositions d'arni de cétohexokinase (khk) et procédés pour les utiliser
WO2015130584A2 (fr) 2014-02-25 2015-09-03 Merck Sharp & Dohme Corp. Adjuvants de vaccins sous forme de nanoparticules lipidiques et systèmes d'administration d'antigènes
WO2015148247A1 (fr) 2014-03-24 2015-10-01 Shire Human Genetic Therapies, Inc. Thérapie basée sur l'arnm pour le traitement des maladies oculaires
WO2015199952A1 (fr) 2014-06-25 2015-12-30 Acuitas Therapeutics Inc. Nouveaux lipides et formulations nanoparticulaires lipidiques pour l'administration d'acides nucléiques
WO2016048903A1 (fr) * 2014-09-22 2016-03-31 Intrexon Corporation Contrôle thérapeutique amélioré de formes hétérodimères et à chaîne unique de l'interleukine-12
WO2017004924A1 (fr) 2015-07-07 2017-01-12 卫小会 Procédé et système de communication pour le partage d'informations de navigation

Non-Patent Citations (138)

* Cited by examiner, † Cited by third party
Title
"Oxford Dictionary Of Biochemistry And Molecular Biology", 2000, OXFORD UNIVERSITY PRESS
"Pharmaceutical Salts: Properties, Selection, and Use", 2008, WILEY-VCH
"Remington: The Science and Practice of Pharmacy", 2005, LIPPINCOTT WILLIAMS & WILKINS
"Remington's Pharmaceutical Sciences", 1985, MACK PUBLISHING COMPANY, pages: 1418
"The Dictionary of Cell and Molecular Biology", 1999, ACADEMIC PRESS
A. R. GENNARO: "Remington: The Science and Practice of Pharmacy", 2006, LIPPINCOTT, WILLIAMS & WILKINS
ABRAHAM ET AL.: "Chaotic Mixer for Microchannels", SCIENCE, vol. 295, 2002, pages 647 - 651
AKINC ET AL., MOL THER., vol. 17, 2009, pages 872 - 879
AKINC ET AL., NAT BIOTECHNOL., vol. 26, 2008, pages 561 - 569
ALEKU ET AL., CANCER RES., vol. 68, 2008, pages 9788 - 9798
ANAND; CHERESH, CURR OPIN HEMATOL, vol. 18, 2011, pages 171 - 176
ANDRE ET AL., CURR GENE THER., vol. 10, 2010, pages 267 - 280
ASSELIN-PATUREL, C. ET AL., CANCER, vol. 91, 2001, pages 113 - 122
BAR M ET AL., STEM CELLS, vol. 26, 2008, pages 2496 - 2505
BARTEL, CELL, vol. 136, 2009, pages 215 - 233
BELLIVEAU ET AL.: "Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA", MOLECULAR THERAPY-NUCLEIC ACIDS, vol. 1, 2012, pages e37, XP002715253, DOI: doi:10.1038/mtna.2012.28
BERGE ET AL., JOURNAL OF PHARMACEUTICAL SCIENCE, vol. 66, 1977, pages 1 - 19
BONAUER ET AL., CURR DRUG TARGETS, vol. 11, 2010, pages 943 - 949
BRIEBA ET AL., BIOCHEMISTRY, vol. 41, 2002, pages 5144 - 5149
CHAKRABARTI R ET AL: "Plasmids encoding membrane-bound IL-4 or IL-12 strongly costimulate DNA vaccination against carcinoembryonic antigen (CEA)", VAC, ELSEVIER, AMSTERDAM, NL, vol. 22, no. 9-10, 12 March 2004 (2004-03-12), pages 1199 - 1205, XP004493383, ISSN: 0264-410X, DOI: 10.1016/J.VACCINE.2003.09.023 *
CHAPPELL ET AL., PNAS, vol. 101, 2004, pages 9590 - 9594
CHEN ET AL.: "Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation", J. AM. CHEM. SOC., vol. 134, no. 16, 2012, pages 6948 - 51, XP002715254, DOI: doi:10.1021/ja301621z
CHENG ET AL., PNAS, vol. 91, 1994, pages 5695 - 5699
CHIARELLA ET AL., CURR GENE THER., vol. 10, 2010, pages 281 - 286
CHUNG ET AL., BMC SYSTEMS BIOLOGY, vol. 6, 2012, pages 134
COMERON; AGUADE, J. MOL. EVOL., vol. 47, 1998, pages 268 - 74
CONTRERAS; RAO, LEUKEMIA, vol. 26, 20 December 2011 (2011-12-20), pages 404 - 413
COUGHLIN, C. M. ET AL., CANCER RES., vol. 57, 1997, pages 2460 - 2467
CUSI, HUM VACCIN., vol. 2, 2006, pages 1 - 7
DATABASE WPI Week 201029, 31 March 2010 Derwent World Patents Index; AN 2010-E33821, XP002783650 *
DE JONGE ET AL., GENE THER., vol. 13, 2006, pages 400 - 411
DEFOUGEROLLES, HUM GENE THER., vol. 19, 2008, pages 125 - 132
DEKOKER ET AL., ADV DRUG DELIV REV., vol. 63, 2011, pages 748 - 761
ENDRES ET AL., BIOMATERIALS, vol. 32, 2011, pages 7721 - 7731
ESVELT ET AL., NATURE, vol. 472, 2011, pages 499 - 503
ESVELT ET AL., NATURE, vol. 472, no. 7344, 2011, pages 499 - 503
FANG ET AL., EXPERT OPIN BIOL THER., vol. 12, 2012, pages 385 - 389
FOTIN-MLECZEK ET AL., J. IMMUNOTHER., vol. 34, 2011, pages 1 - 15
FOX; ERIL, DNA RES., vol. 17, no. 3, 2010, pages 185 - 96
FROST, EXPERT OPIN. DRUG DELIV, vol. 4, 2007, pages 427 - 440
FULLER ET AL., BIOMATERIALS, vol. 29, 2008, pages 1526 - 1532
GARDIN ET AL., ELIFE, vol. 3, 2014, pages e03735
GATELY, MK ET AL., ANNU REV IMMUNOL., vol. 16, 1998, pages 495 - 521
GENTNER; NALDINI, TISSUE ANTIGENS, vol. 80, 2012, pages 393 - 403
GODFRIN ET AL., EXPERT OPIN BIOL THER., vol. 12, 2012, pages 127 - 133
GOFF LA ET AL., PLOS ONE, vol. 4, 2009, pages e7192
GRIMSON A; FARH KK; JOHNSTON WK; GARRETT-ENGELE P; LIM LP; BARTEL DP, MOL CELL, vol. 27, no. 1, 6 July 2007 (2007-07-06), pages 91 - 105
GUSTAFSSON ET AL., JOURNAL/TRENDS BIOTECHNOL, vol. 22, 2004, pages 346 - 53
GUSTAFSSON ET AL., PROTEIN EXPRESSION AND PURIFICATION, vol. 83, 2012, pages 37 - 46
GUTBIER ET AL., PULM PHARMACOL. THER., vol. 23, 2010, pages 334 - 344
HERBST ET AL., J CLIN ONCOL, vol. 31, 2013, pages 3000
HOJMAN, CURR GENE THER., vol. 10, 2010, pages 128 - 138
HU ET AL., PROC NATL ACAD SCI USA., vol. 108, 2011, pages 10980 - 10985
HUCKRIEDE ET AL., J LIPOSOME RES., vol. 17, 2007, pages 39 - 47
IKEDA; RICHARDSON, PROC NATL ACAD SCI, vol. 83, 1986, pages 3614 - 3618
IKEMURA, J. MOL. BIOL., vol. 151, no. 3, 1981, pages 389 - 409
INGOLIA ET AL., SCIENCE, vol. 324, no. 5924, 2009, pages 218 - 23
J POUTOU ET AL: "Safety and antitumor effect of oncolytic and helper-dependent adenoviruses expressing interleukin-12 variants in a hamster pancreatic cancer model", GENE THERAPY, vol. 22, no. 9, 4 May 2015 (2015-05-04), GB, pages 696 - 706, XP055498024, ISSN: 0969-7128, DOI: 10.1038/gt.2015.45 *
JIMA DD ET AL., BLOOD, vol. 116, 2010, pages ell8 - el27
JUNJIE LI ET AL., CURRENT BIOLOGY, vol. 15, 23 August 2005 (2005-08-23), pages 1501 - 1507
JUO, PEI-SHOW: "Concise Dictionary of Biomedicine and Molecular Biology", 2002, CRC PRESS
KANG, W. K. ET AL., HUMAN GENE THERAPY, vol. 12, 2001, pages 671 - 684
KAUFMANN ET AL., MICROVASC RES, vol. 80, 2010, pages 286 - 293
KEDDE ET AL.: "A Pumilio-induced RNA structure switch in p27-3'UTR controls miR-221 and miR-22 accessibility", NATURE CELL BIOLOGY, 2010
KHLEIF, PROCEEDINGS FROM THE EUROPEAN CANCER CONGRESS 2013, 27 September 2013 (2013-09-27)
KIM ET AL., NATURE, vol. 351, no. 6324, 1991, pages 331 - 2
KORE ET AL., BIOORGANIC & MEDICINAL CHEMISTRY, vol. 21, 2013, pages 4570 - 4574
KUPPUSAMY KT ET AL., CURR. MOL MED, vol. 13, no. 5, 2013, pages 757 - 764
LANDGRAF ET AL., CELL, vol. 129, 2007, pages 1401 - 1414
LASEK W. ET AL., CANCER IMMUNOL IMMUNOTHER, vol. 63, 2014, pages 419 - 435,424
LEACH ET AL., J. AM. CHEM. SOC., vol. 114, 1992, pages 3675 - 3683
LEONARD, J. P. ET AL., BLOOD, vol. 90, 1997, pages 2541 - 2548
LEONARD, J.P. ET AL., BLOOD, vol. 90, 1997, pages 2541 - 2548
LIU; HUANG, MOLECULAR THERAPY, 2010, pages 669 - 670
LOVE ET AL., PROC NATL ACAD SCI USA., vol. 107, 2010, pages 1864 - 1869
LUND ET AL., PHARM RES., vol. 27, 2010, pages 400 - 420
MAHON ET AL., BIOCONJUG. CHEM., vol. 21, 2010, pages 1448 - 1454
MANDAL; ROSSI, NAT. PROTOC., vol. 8, no. 3, 2013, pages 568 - 82
MANTSCH ET AL., BIOCHEM., vol. 14, 1993, pages 5593 - 5601
MATSUDA ET AL., PLOS ONE, vol. 11, no. 5, 2010, pages el5057
MATSUDA; MAURO, PLOS ONE, vol. 5, 2010, pages 11
MATSUDA; MAURO: "describing masking agents LNA polynucleotides and EJCs", PLOS ONE, vol. 5, 2010, pages 11
MEIJER HA ET AL., SCIENCE, vol. 340, 2013, pages 82 - 85
MELLMAN ET AL., NATURE, vol. 480, 2011, pages 480 - 489
MI-HUA TAO ET AL: "Membrane-bound interleukin 12 induced stronger antitumor immunity than soluble interleukin 12 without inducing circulating interferon [gamma]", CANCER RESEARCH, vol. 65, no. 9, 1 May 2005 (2005-05-01), US, pages 1410 - -, XP055514162, ISSN: 0008-5472 *
MORIN RD ET AL., GENOME RES, vol. 18, 2008, pages 610 - 621
MURUGAIAH ET AL., ANALYTICAL BIOCHEMISTRY, vol. 401, 2010, pages 61
NASERI ET AL.: "Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: Structure, Preparation and Application", ADV. PHARM. BULL., vol. 5, 2015, pages 305 - 13
NASTALA, C. L. ET AL., J IMMUNOL, vol. 153, 1994, pages 1697 - 1706
NEWMAN; BETTINGER, GENE THER., vol. 14, 2007, pages 465 - 475
NORBURY: "Nature Reviews Molecular Cell Biology", 29 August 2013, AOP, article "Cytoplasmic RNA: a case of the tail wagging the dog"
PAN, W-Y. ET AL., MOL. THER., vol. 20, no. 5, 2012, pages 927 - 937
PASCOLO, EXPERT OPIN. BIOL. THER., vol. 4, pages 1285 - 1294
PEER ET AL., PROC NATL ACAD SCI USA, vol. 104, June 2007 (2007-06-01), pages 4095 - 4100
PICCIRILLI ET AL., NATURE, vol. 343, 1990, pages 33 - 37
POSTEMA; GILJA, CURR PHARM BIOTECHNOL., vol. 8, 2007, pages 355 - 361
QUERIDO ET AL., J. CELL SCI., vol. 124, 2014, pages 1703 - 1714
RODRIGUEZ ET AL., SCIENCE, vol. 339, 2013, pages 971 - 975
ROSENBERG ET AL., SCIENCE, vol. 233, no. 4770, 1986, pages 1318 - 21
SACCO, S. ET AL., BLOOD, vol. 90, 1997, pages 4473 - 4479
SANDHU ET AL., IN SILICO BIOL., vol. 8, no. 2, 2008, pages 187 - 92
SANTEL ET AL., GENE THER, vol. 13, 2006, pages 1222 - 1234
SANTEL ET AL., GENE THER, vol. 13, 2006, pages 1360 - 1370
SAUDEMONT, A. ET AL., LEUKEMIA, vol. 16, 2002, pages 1637 - 1644
SCHROEDER ET AL., J INTERN MED., vol. 267, 2010, pages 9 - 21
SHARP; LI, NUCLEIC ACIDS RES., vol. 15, no. 3, 1987, pages 1281 - 95
SIEGWART ET AL., PROC NATL ACAD SCI USA, vol. 108, 2011, pages 12996 - 13001
SIEGWART ET AL., PROC NATL ACAD SCI USA., vol. 108, 2011, pages 12996 - 3001
SILVA ET AL.: "Delivery Systems for Biopharmaceuticals. Part I: Nanoparticles and Microparticles", CURR. PHARM. TECHNOL., vol. 16, 2015, pages 940 - 954
SILVA ET AL.: "Lipid nanoparticles for the delivery of biopharmaceuticals", CURR. PHARM. BIOTECHNOL., vol. 16, 2015, pages 291 - 302
SONG ET AL., NATURE BIOTECHNOL., vol. 23, 2005, pages 709 - 717
STRUMBERG ET AL., INT J CLIN PHARMACOL THER, vol. 50, 2012, pages 76 - 78
SU ET AL., MOL PHARM., vol. 8, no. 3, 6 June 2011 (2011-06-06), pages 774 - 87
SUZUKI ET AL., DNA RES., vol. 15, no. 6, 2008, pages 357 - 65
SWITZER ET AL., BIOCHEMISTRY, vol. 32, 1993, pages 10489 - 10496
TOR ET AL., J. AM. CHEM. SOC., vol. 115, 1993, pages 4461 - 4467
TOURIOL ET AL., BIOLOGY OF THE CELL, vol. 95, 2003, pages 169 - 178
VAZ C ET AL., BMC GENOMICS, vol. 11, 2010, pages 288
VIDIGAL JA; VENTURA A, SEMIN CANCER BIOL., vol. 22, no. 5-6, 2012, pages 428 - 436
VILLALOBOS ET AL., JOURNAL/BMC BIOINFORMATICS, vol. 7, 2006, pages 285
VOELLENKLE C ET AL., RNA, vol. 18, 2012, pages 472 - 484
WANG ET AL., CANCER IMMUNOL RES., vol. 2, no. 9, 2014, pages 846 - 56
WANG ET AL., NAT MATER., vol. 5, 2006, pages 791 - 796
WANG ET AL.: "Delivery of oligonucleotides with lipid nanoparticles", ADV. DRUG DELIV. REV., vol. 87, 2015, pages 68 - 80
WEIDE ET AL., J IMMUNOTHER., vol. 31, 2008, pages 180 - 188
WEIDE ET AL., J IMMUNOTHER., vol. 32, 2009, pages 498 - 507
WELCH ET AL., PLOS ONE, vol. 4, no. 9, 2009, pages e7002
WEN-YU PAN ET AL., MOL THERAP, vol. 20, no. 5, May 2012 (2012-05-01), pages 927 - 937
WEN-YU PAN ET AL: "Cancer Immunotherapy Using a Membrane-bound Interleukin-12 With B7-1 Transmembrane and Cytoplasmic Domains", MOLECULAR THERAPY : THE JOURNAL OF THE AMERICAN SOCIETY OF GENE THERAPY, vol. 20, no. 5, 1 May 2012 (2012-05-01), US, pages 927 - 937, XP055327744, ISSN: 1525-0016, DOI: 10.1038/mt.2012.10 *
WHITESIDES, GEORGE M.: "The Origins and the Future of Microfluidics", NATURE, vol. 442, 2006, pages 368 - 373, XP055123139, DOI: doi:10.1038/nature05058
XI YANG ET AL: "Mouse interleukin-12/FasTI: A novel bi-functional fusion protein for cancer immuno/gene therapy", INTERNATIONAL JOURNAL OF ONCOLOGY, vol. 48, no. 6, 7 April 2016 (2016-04-07), GR, pages 2381 - 2386, XP055498023, ISSN: 1019-6439, DOI: 10.3892/ijo.2016.3475 *
YAKUBOV ET AL., BIOCHEM. BIOPHYS. RES. COMMUN., vol. 394, no. 1, 2010, pages 189 - 193
YOO JK ET AL., STEM CELLS DEV., vol. 21, no. 11, 2012, pages 2049 - 2057
YOON; PARK, EXPERT OPIN DRUG DELIV., vol. 7, 2010, pages 321 - 330
ZANDBERG ET AL., ORAL ONCOL., vol. 50, 2014, pages 627 - 632
ZANGI ET AL., NATURE BIOTECHNOLOGY, 2013
ZHIGALTSEVET: "Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing", LANGMUIR, vol. 28, 2012, pages 3633 - 40, XP055150435, DOI: doi:10.1021/la204833h
ZHU ET AL., NUCLEIC ACIDS RESEARCH, 2013

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3523323A4 (fr) * 2016-10-07 2020-06-17 Board of Regents, The University of Texas System Lymphocytes t exprimant il-12 ancrée sur membrane pour le traitement du cancer
US11421010B2 (en) 2016-10-07 2022-08-23 Board Of Regents, The University Of Texas System T cells expressing membrane-anchored IL-12 for the treatment of cancer
US11446332B2 (en) 2017-04-13 2022-09-20 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
US11655277B2 (en) 2018-10-03 2023-05-23 Xencor, Inc. IL-12 heterodimeric Fc-fusion proteins
US11358999B2 (en) 2018-10-03 2022-06-14 Xencor, Inc. IL-12 heterodimeric Fc-fusion proteins
US10993967B2 (en) 2018-10-17 2021-05-04 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
US11419898B2 (en) 2018-10-17 2022-08-23 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
WO2020123716A1 (fr) 2018-12-11 2020-06-18 Obsidian Therapeutics, Inc. Il12 liée à la membrane, compositions et procédés de régulation accordable
WO2020160350A1 (fr) * 2019-02-01 2020-08-06 Board Of Regents, The University Of Texas System Thérapie par lymphocytes t à il-12 modifiés pour le traitement du cancer
CN110095463A (zh) * 2019-03-15 2019-08-06 中国人民解放军陆军军医大学第二附属医院 一种乳糜定性检测的试剂盒
WO2020252404A1 (fr) 2019-06-12 2020-12-17 Obsidian Therapeutics, Inc. Compositions de ca2 et procédés de régulation accordable
US11058725B2 (en) 2019-09-10 2021-07-13 Obsidian Therapeutics, Inc. CA2 compositions and methods for tunable regulation
US11851466B2 (en) 2019-10-03 2023-12-26 Xencor, Inc. Targeted IL-12 heterodimeric Fc-fusion proteins
WO2021072172A1 (fr) * 2019-10-09 2021-04-15 Translate Bio, Inc. Compositions, méthodes et utilisations d'arn messager
CN114728176A (zh) * 2019-10-09 2022-07-08 川斯勒佰尔公司 信使rna的组合物、方法和使用
WO2021221127A1 (fr) * 2020-04-30 2021-11-04 Vlp Therapeutics, Inc. Immunothérapie à cytokine
WO2021262985A1 (fr) * 2020-06-25 2021-12-30 Amunix Pharmaceuticals, Inc. Conjugués de cytokine
WO2022020811A1 (fr) * 2020-07-24 2022-01-27 Strand Therapeutics, Inc. Nanoparticule de nanoparticule lipidique comprenant des nucléotides modifiés
US11661459B2 (en) 2020-12-03 2023-05-30 Century Therapeutics, Inc. Artificial cell death polypeptide for chimeric antigen receptor and uses thereof
US11883432B2 (en) 2020-12-18 2024-01-30 Century Therapeutics, Inc. Chimeric antigen receptor system with adaptable receptor specificity
WO2023196988A1 (fr) * 2022-04-07 2023-10-12 Modernatx, Inc. Procédés d'utilisation d'arnm codant pour il-12

Also Published As

Publication number Publication date
JP2020520640A (ja) 2020-07-16
AU2018270111A1 (en) 2019-11-28
US11873327B2 (en) 2024-01-16
AU2018270111B2 (en) 2022-07-14
JP7285220B2 (ja) 2023-06-01
EP3625246A1 (fr) 2020-03-25
JP2023059880A (ja) 2023-04-27
CA3063723A1 (fr) 2018-11-22
US20200102363A1 (en) 2020-04-02
US11421011B2 (en) 2022-08-23
US20230116843A1 (en) 2023-04-13

Similar Documents

Publication Publication Date Title
AU2018270111B2 (en) Polynucleotides encoding tethered interleukin-12 (IL12) polypeptides and uses thereof
US11571463B2 (en) Polynucleotides encoding interleukin-12 (IL12) and uses thereof
US11649461B2 (en) Polynucleotides encoding α-galactosidase A for the treatment of Fabry disease
US20220071915A1 (en) Polynucleotides encoding citrin for the treatment of citrullinemia type 2
US10426738B2 (en) Polynucleotides encoding methylmalonyl-CoA mutase
US11001861B2 (en) Polynucleotides encoding galactose-1-phosphate uridylyltransferase for the treatment of galactosemia type 1
US20190298657A1 (en) Polynucleotides Encoding Acyl-CoA Dehydrogenase, Very Long-Chain for the Treatment of Very Long-Chain Acyl-CoA Dehydrogenase Deficiency
EP3458107A1 (fr) Polynucléotides codant pour jagged1 pour le traitement du syndrome d'alagille

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18729295

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3063723

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2019563454

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2018270111

Country of ref document: AU

Date of ref document: 20180518

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2018729295

Country of ref document: EP

Effective date: 20191218